Inhibitors of crispr-cas9

ABSTRACT

Cas9-inhibiting polypeptide compositions and methods are provided.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of priority to U.S. Provisional Patent Application No. 62/422,850, filed Nov. 16, 2016, which is incorporated by referenced for all purposes.

BACKGROUND OF THE INVENTION

The ability to prevent attack from viruses is a hallmark of cellular life. Bacteria employ multiple mechanisms to resist infection by bacterial viruses (phages), including restriction enzymes and CRISPR-Cas systems (Labrie, S. J., Samson, J. E., and Mineau, S. (2010). Nat Rev Micro, 8, 317-327). CRISPR arrays possess the sequence-specific remnants of previous encounters with mobile genetic elements as small spacer sequences located between their clustered regularly interspaced short palindromic repeats (Mojica, F. J. M et al. (2005). J. Mol. Evol., 60, 174-182). These spacers are utilized to generate guide RNAs that facilitate the binding and cleavage of a programmed target (Brouns, S. J. J et al. (2008). Science, 321, 960-964; Garneau, J. E. et al. (2010). Nature, 468, 67-71). CRISPR-associated (cas) genes that are required for immune function are often found adjacent to the CRISPR array (Marraffini, L. A. (2015). CRISPR-Cas immunity in prokaryotes, Nature, 526, 55-61; Wright, A. V., Nuñez, J. K., and Doudna, J. A. (2016). Cell, 164, 29-44). Cas proteins not only carry out the destruction of a foreign genome (Garneau, J. E. et al, (2010). Nature, 468, 67-71), but also facilitate the production of mature CRISPR RNAs (crRNAs) (Deltcheva; Haurwitz, R. E et al. (2010). Science, 329, 1355-1358) and the acquisition of foreign sequences into the CRISPR array (Nuñez, J. K. et al. (2014). Nat. Struct. Mol. Biol, 21, 528-534; Yosef, I., Goren, M. G., and Qimron, U. (2012). Nucleic Acids Research, 40, 5569-5576).

CRISPR-Cas adaptive immune systems are common and diverse in the bacterial world. Six different types (I-VI) have been identified across bacterial genomes (Abudayyeh, O. O et al. (2016). Science aaf5573; Makarova., K. S. et al. (2015). Nat Rev Micro, 13, 722-736). Nat Rev Micro, 13, 722-736), with the ability to cleave target DNA or RNA sequences as specified by the RNA guide. The facile programmability of CRISPR-Cas systems has been widely exploited, opening up the door to many novel genetic technologies (Barrangou, R., and Doudna, J. A. (2016), Nature Biotechnology, 34, 933-941). Most of these technologies use Cas9 from Streptococcus pyogenes (Spy), together with an engineered single guide RNA as the foundation for such applications, including gene editing in animal cells (Cong, L. et al. (2013). Science 339, 819-823; Jinek, M. et al. (2012). Science, 337, 816-821; Mali, P, et al. (2013). Science, 339, 823-826; Qi, L. S. et al. (2013). Cell, 152, 1173-1183). Additionally, Cas9 orthologs within the II-A subtype have been investigated for gene editing applications (Ran, F. A. et al. (2015). Nature 520, 186-191), and new Class 2 CRISPR single protein effectors such as Cpf1 (Type V (Zetsche, B. et al. (2015). Cell, 163, 759-771)) and C2c2 (Type VI (Abudayyeh, O. O et al. (2016). Science aaf5573; East-Seletsky, A. et al. (2016). Nature 538, 270-273) are being characterized. Class 1 CRISPR-Cas systems (Type I, III, and IV) are RNA-guided multi-protein complexes and thus have been overlooked for most genomic applications due to their complexity. These systems are, however, the most common in nature being found in nearly half of all bacteria and 85% of archaea (Makarova, K. S. et al. (2015). Nat Rev Micro, 13, 722-736). Nat Rev Micro, 13, 722-736).

In response to the bacterial war on phage infection, phages, in turn, often encode inhibitors of bacterial immune systems that enhance their ability to lyse their host bacterium or integrate into its genome (Samson, J. E. et al. (2013). Nat Rev Micro, 11, 675-687). The first examples of phage-encoded “anti-CRISPR” proteins came for the (Class 1) type I-E and I-F systems in Pseudomonas aeruginosa (Bondy-Denomy et al. (2013). Nature, 493, 429-432; Pawluk, A. et al. (2014). mBio 5, e00896). Remarkably, ten type I-F anti-CRISPR and four type I-E anti-CRISPR genes have been discovered to date (Pawluk, A. et al. (2016). Nature Microbiology, 1, 1-6), all of which encode distinct, small proteins (50-150 amino acids), previously of unknown function. Our biochemical investigation of four I-F anti-CRISPR proteins revealed that they directly interact with different Cas proteins in the multi-protein CRISPR-Cas complex to prevent either the recognition or cleavage of target DNA (Bondy-Denomy, J et al. (2015). Nature, 526, 136-139). Each protein has a distinct sequence, structure, and mode of action (Maxwell, K. L. et al. (2016). Nature Communications, 7, 13134; Wang, X. (2016). Nat. Struct. Biol 23, 868-870). These findings support the independent evolution of CRISPR-Cas inhibitors and suggests that many more are yet to be discovered. In this light, a recent paper utilized the conservation of signature anti-CRISPR associated (aca) gene with a predicted helix-turn-helix (HTH) motif to identify anti-CRISPR genes outside of P. aeruginosa. This led to the authors finding anti-CRISPRs across proteobacteria, broadly spanning the type I-F CRISPR-Cas phylogeny (Pawluk, A. et al, (2016). Nature Microbiology, 1,1-6). This suggests that anti-CRISPRs may exist for all CRISPR systems, with methods needed to enable their discovery.

The type I anti-CRISPRs in P. aeruginosa are expressed from integrated phage genomes (prophages), leading to the constitutive inactivation of the host CRISPR-Cas system (Bondy-Denomy et al. (2013). Nature, 493,429-432. This can often lead to a situation where a prophage possesses a DNA target with perfect identity to a co-occurring CRISPR spacer in the same cell, called “self-targeting” (FIG. 1A). This situation makes CRISPR-Cas inactivation a requirement for survival, as in the absence of prophage anti-CRISPR genes, the host genome is cleaved in the act of targeting the prophage (Bondy-Denomy et al. (2013). Nature 493, 429-432; Edgar, R., and Qimron, U. (2010). J. Bacteriol 192, 6291-6294). Expression of an anti-CRISPR neutralizes this risk, however, allowing lysogen survival. We surmised that genomes possessing a CRISPR system with apparent self-targeting would be candidates for the identification of new CRISPR-Cas inhibitors. Here, we describe the identification of previously unknown phage-encoded CRISPR-Cas9 inhibitors in Listeria monocytogenes using a bioinformatics approach to identify incidents of self-targeting. We show that two of these inhibitors can also block the activity of S. pyogenes Cas9 in bacterial and human cells.

DEFINITIONS

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The term “gene” means the segment of DNA involved in producing a polypeptide chain. ft may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter.

An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

“Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. In some cases, conservatively modified variants of Cas9 or sgRNA can have an increased stability, assembly, or activity as described herein.

The following eight groups each contain amino acids that are conservative substitutions for one another:

-   1) Alanine (A), Glycine (G); -   2) Aspartic acid (D), Glutamic acid (E); -   3) Asparagine (N), Glutamine (Q); -   4) Arginine (R), Lysine (K); -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); -   7) Serine (S), Threonine (T); and -   8) Cysteine (C), Methionine (M)     (see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y.     (1984)).

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.

As used in herein, the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which are described in Altschul el al., (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them, The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. :For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The “CRISPR/Cas” system refers to a class of bacterial systems for defense against foreign nucleic acid. CRISPR/Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR/Cas systems include type II, III, V, and VI sub-types. Wild-type type II CRISPR/Cas systems utilize the RNA-mediated nuclease, Cas9 in complex with guide and activating RNA to recognize and cleave foreign nucleic acid.

Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydia-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firniicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 polyeptide is the Streptococcus pyogenes Cas9 polyeptide. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737 ; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Sampson et al., Nature. 2013 May 9; 497(7448):254-7; and Jinek, et al., Science, 2012 Aug. 17; 337(6096):816-21. The Cas9 protein can be nuclease defective. For example, the Cas9 protein can be a nicking endonuclease that nicks target DNA, but does not cause double strand breakage. Cas9 can also have both nuclease domains deactivated to generate “dead Cas9” (dCas9), a programmable DNA-binding protein with no nuclease activity. In some embodiments, dCas9 DNA-binding is inhibited by the polypeptides described herein.

BRIEF SUMMARY OF THE INVENTION

In one aspect, methods of inhibiting a Cas9 polypeptide in a cell are provided. In some embodiments, the method comprises. introducing a Cas9-inhibiting polypeptide into a cell, wherein: the Cas9-inhibiting polypeptide is heterologous to the cell, and the Cas9-inhibiting polypeptide is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%) identical to any one or more of SEQ ID NO: 1-170, thereby inhibiting the Cas9 polypeptide in a cell. In sonic embodiments, the method comprises contacting the Cas9 inhibiting polypeptide with a Cas9 polypeptide in the cell. In some embodiments, the method comprises contacting the Cas9 inhibiting polypeptide with other components of the CRISPR-Cas9 system in the cell, thereby indirectly inhibiting Cas9 polypeptide activity.

In some embodiments, the Cas9-inhibiting polypeptide comprises one of SEQ ID NO: 1-170.

In some embodiments, the cell comprises the Cas9 polypeptide before the introducing. In some embodiments, the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding the Cas9 polypeptide. In some embodiments, the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell prior to the introducing.

In some embodiments, the cell comprises the Cas9 polypeptide after the introducing. In some embodiments, the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell after to the introducing.

In some embodiments, the introducing comprises expressing the Cas9-inhibiting polypeptide in the cell from an expression cassette that is present in the cell and heterologous to the cell, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, the promoter is an inducible promoter and the introducing comprises contacting the cell with an agent that induces expression of the Cas9-inhibiting polypeptide.

In some embodiments, the introducing comprises introducing an RNA encoding the Cas9-inhibiting polypeptide into the cell and expressing the Cas9-inhibiting polypeptide in the cell from the RNA.

In some embodiments, the introducing comprises inserting the Cas9-inhibiting polypeptide into the cell or contacting the cell with the Cas9-inhibiting polypeptide.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a blood or an induced pluripotent stem cell. In some embodiments, the cell is a prokaryotic cell.

In some embodiments, the method occurs ex: vivo. In some embodiments, the cells are introduced into a mammal after the introducing and contacting. In some embodiments, the cells are autologous to the mammal.

Also provided is a cell (optionally isolated) comprising a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is heterologous to the cell and the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO: 1-170. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell In some embodiments, the cell is a prokaryotic cell.

Also provided is a polynucleotide comprising a nucleic acid encoding a Cas9-inhibiting polypeptide. In some embodiments, the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO: 1-170. in some embodiments, the polynucleotide comprises an expression cassette, the expression cassette comprising a promoter operably linked to the nucleic acid. In some embodiments, the promoter is heterologous to the polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, the promoter is inducible. In some embodiments, the polynucleotide is DNA or RNA.

Also provided is a vector comprising the expression cassette as described above or elsewhere herein. In some embodiments, the vector is a viral vector.

Also provided is a (optionally isolated) Cas9-inhibiting polypeptide. In some embodiments, the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ NO:1-170.

Also provided is a pharmaceutical composition comprising the polynucleotide or the polypeptide as described above or elsewhere herein.

Also provided is a delivery vehicle comprising the polynucleotide or the polypeptide as described above or elsewhere herein. In some embodiments, the delivery vehicle is a liposome or nanoparticle.

Other aspects are described in the remainder of this document.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. A survey for CRISPR-Cas9 genomic self-targeting (ST) in Listeria monocytogenes.

FIG. 1A A schematic depicting the principle of genomic self-targeting, where a mobile genetic element (MGE) possesses a target sequence for a spacer in a CRISPR array in the same genome. CRISPR-Cas9 function in this “self-targeting genome” is presumably inactive for continued cell viability.

FIG. 1B The abundance of genomes with cas9-linked self-targeting (red) and those without ST (grey), in L. monocytogenes genomes.

FIG. 1C An example of an ST event, where spacer 16 in the CRISPR array of strain J0161 has a perfect PAM and protospacer match with a resident prophage (φJ0161a) (spacer sequences: crRNA (SEQ ID NO:171), protospacer and protospacer complement (SEQ ID NOS:172-173)).

FIG. 2A-D. A prophage from L. monocytogenes J0161 contains two CRISPR-Cas9 inhibitor genes.

FIG. 2A The type II-A CRISPR-Cas locus in L. monocytogenes 10403s. Four cas genes are indicated, along with tracrRNA and CRISPR array, containing 30 spacers. The predicted direction of transcription is indicated with black arrows. Subsequent experiments utilize a non-targeted plasmid (pNT) and a targeted plasmid (pT) that has a protospacer matching spacer 1 in this strain.

FIG. 2B Representative pictures of transformed colonies after CRISPR-Cas-targeted (pT) or non-targeted (pNT) plasmids were electroporated into phage-cured (φcure) strains of L. monocytogenes 10403s and wild type (wt) J0161 (contains the φJ0161a prophage) shown in red to denote self-targeting. Analyzed φcure 10403s variants include a cas9-deletion strain (Acas9), a lysogen of φJ0161a (::φJ0161a), strains constitutively expressing individual CRISPR-Cas9 inhibitor genes from φJ0161a (+acrIIA1, +acrIIA2) and a lysogen of φJ0161a with CRISPR-Cas9 inhibitor genes deleted (::φJ0161aΔacrIIA1-2). See FIG. 7 for a comparison of wt and φcure 10403s.

FIG. 2C 10403s φcure and wt J0161 strains were assessed for transformation efficiency. The 10403s φcure cas9-deletion strain (Acas9), constitutively expressed cas9 (Δcas9+cas9) and φJ0161a lysogens of these strains (::φJ0161a) were analyzed. Error bars reflect the standard deviation of three biological replicates. L.D. limit of detection.

FIG. 2D Comparison of the open reading frames from two similar prophages from L. monocytogenes 10403s and J0161. Unique genes (red) comprising ten fragments of φJ0161 were tested for CRISPR-Cas9 inhibition in 10403s. n.e., No effect on CRISPR-Cas9 activity, tox., fragment toxic when expressed, t., location of self-targeted protospacer. The encircled fragment exhibited anti-CRISPR activity with two genes (acrAII1, acrAII2) independently capable of inhibiting CRISPR-Cas activity. Conserved (grey) genes were not tested. For reference, phage genes involved in cell lysis, capsid assembly and host integration (int.) are labeled.

FIG. 3A-C. Genomic organization and prevalence of acrII4 genes

FIG. 3A The genomic context of actIIA1 (1) and its homolog from L. monocytogenes (orfD) are depicted to scale as cartoons with acIIA1 homologs in vertical alignment. Typically, acrIIA genes are encoded within prophages adjacent to or near the phage lysin. (ply) gene. Genomic neighbors of acrIIA1 and orfD (acrIIA1-4, orfA-E) are shown. Individual genes (***) were assayed for CRISPR-Cas9 inhibition in L. monocytogenes 10403s (see FIG. 9). Helix-turn-helix (HUI) and AP2 DNA binding motifs were detected in some proteins using hidden markov model (HMM) prediction software (Soding, J., Siegert, A., and Lupas, A. N. (2005). Nucleic Acids Research, 33, W244-W248).

FIG. 3B Pie-graph representation of the frequency of each acrIIA gene co-occurrences

FIG. 3C Pie-graph representation of the prevalence of acrIIA and cas9 genes in the L. monocytogenes pan-genome. See Supplementary Table 1 for relevant accession numbers.

FIG. 4A. Phylogenetic analysis of AcrIIA1-4 homologs

A phylogenetic reconstruction of full-length protein sequences identified following an iterative psi-BLASTp search to query all non-redundant protein sequences within GenBank for (FIG. 4A)

BLASTp was used to construct a similar tree for (FIG. 4B) AcrIIA2, (FIG. 4C) AcrIIA3, and FIG. 4D AcrIIA4 (see Methods). Selected bootstrapping support values are denoted with filled ovals (≥90%), open rectangles (≥70%) or dashed lines (<70%). The sequence family that is boxed-in represents the family that was tested for anti-CR1SPR function. Other homologs reflect distinct sequence families present in the genotnes described under the tree.

FIG. 5A-D. Inhibition of Streptococcus pyogenes dCas9 and Cas9.

FIG. 5A A schematic outlining the experimental setup, where single-cell fluorescence of E. coli BW25113 with Strepotococcus pyogenes (Spy) dCas9 and a guide RNA targeted towards a chromosomal red fluorescent protein (RFR) gene was measured.

FIG. 5B Candidate (orf) and validated (act) acrIIA genes were tested for their ability to inhibit dCas9-based repression. Measurements taken reflect the median REP fluorescence value of a single cell in a unimodal population normalized for each candidate gene to a guide RNA-free control. Error bars represent the standard deviation of at least three biological replicates. See FIG. 2 and FIG. 9 for gene-identification information.

FIG. 5C A schematic outlining the experimental setup, where HEK293T cells with a doxycycline-inducible eGFP cassette were transfected with a plasmid encoding a single transcript tracrRNA/eGFP-targeting guide RNA and NLS-SpyCas9 alongside expression constructs encoding one of five codon-optimized phage genes at different ratios. The percent of eGFP positive cells was measured 12 hours after induction by flow cytometry.

FIG. 5D Average percent of eGFP positive cells is depicted+/−standard deviation across biological triplicates. An increasing amount of inhibitor plasmid (in ng) was added from left to right, at a ratio to the Cas9/sgRNA plasmid of 1:1 and 3:1. Data were normalized to transfection with no phage ORF as the baseline.

FIG. 6 is a cartoon depiction of AcrIIA-inhibition of Cas9.

FIG. 7A-F. Self-Targeting by CRISPR-Cas9 in Listeria monocytogenes J0161 is Not Associated with Loss-of-Function Mutations, Related to FIG. 1

FIG. 7A Comparison of type II-A CRISPR-cas loci from Streptococcus pyogenes SF370 (Spy_SF370), Listeria monocytogenes 10403s (Lmo_10403s), Listeria monocytogenes J0161 (Lmo_J0161) and Listeria innocua (Lin_Clip11262). Percent identity between Cas9 protein sequences is shown.

FIG. 7B The CRISPR array of self-targeting strain Lmo J0161. A type II-A CRISPR array, predicted by the CRISPRDetect web utility is shown (Spacer_Sequence: SEQ ID NOS:174-192, respectively; Repeat_Sequence: SEQ ID NO:193). The self-targeting spacer (number 16 (SEQ ID NO:189) is boxed. In bold, are the RNA-coding nucleotides responsible for target recognition.

FIG. 7C A modified CRISPRtarget output, depicting self-targeting by L. monocytogenes J0161. The predicted crRNA processing site is identified by the wedge icon. (sequences: crRNA (SEQ ID NO:194), target DNA and target DNA complement (SEQ ID NOS:195-196)).

FIG. 7D Alignment of tracrRNA loci. (Spy_SF370 (SEQ ID NO:197); Lmo_10403s (SEQ ID NO:198); Lmo_J0161 (SEQ ID NO:199): Lin_Clip11262 (SEQ ID NO:200).

FIG. 7E Alignment of CRISPR loci. (Spy SF370 (SEQ ID NO:201); Lmo_10403s (SEQ ID NO:202); Lmo_J0161 (SEQ ID NO:203): Lin_Clip11262 (SEQ ID NO:204).

FIG. 7F Alignment of Cas9 protein sequences. Residues with essential chemical functionalities are boxed. (Spy_SF370 (SEQ ID NO:205); Lmo_10403s (SEQ ID NO:206); Lmo_J0161 (SEQ ID NO:207): Lin_Clip11262 (SEQ ID NO:208).

FIG. 8. The φ10403s Prophage Does Not Influence Plasmid Targeting in L. monocytogenes 10403s, Related to FIG. 2A-D. Representative plates depicting colonies after transformation and selection for targeted (pT; pRAU31) or non-targeted (pNT; pRAU29) plasmids. Wild type (wt) and nonlysogenic (φcure) strains of 10403s were analyzed. See Table S1 for additional information pertinent to plasmid and strain design and nomenclature.

FIG. 9. Fragments of the φJ0161a Prophage that were Screened for CRISPR-Cas9 Inhibition Activity in L. monocytogenes 10403s, Related to FIG. 2. Representative plates depicting colonies after transformation and selection for targeted (pT; pRAU31) or non-targeted (pNT, pRAU29) plasmids. DNA sequence information is provided for all phage fragments as they are named in FIG. 2d . See Table 51 for additional information pertinent to plasmid and strain design and nomenclature.

FIG. 10A-B. Individual Genes that were Screened for CRISPR-Cas9 Inhibition Activity in L. monocytogenes 10403s, Related to FIG. 2B and FIG. 3A. Representative plates depicting colonies after transformation and selection for targeted (pT; pRAU31) or non-targeted (pNT; pRAU29) plasmids. Given names, locus tags, accession numbers and. DNA sequence information is provided for all candidate type II-A CRISPR-Cas inhibitors. See Table S1 for additional information pertinent to plasmid and strain design and nomenclature.

FIG. 11A-B. Toxicity of an AcrIIA3 Homolog from S. pyogenes in E. coli, Related to FIG. 5.

FIG. 11A Distribution of single-cell REP fluorescence values for E. coli CRISPRi reporter strains with and without expression of AcrIIA proteins. Expression of AcrIIA proteins leads to unimodal shift in population fluorescence towards the sgRNA (no CRISPRi knockdown) state, indicating a uniform disruption of CRISPRi activity. Strains were grown for 2.5 hr in the presence of IPTG to induce CRISPRi, with or without expression of the AcrIIA inhibitor.

FIG. 11B Expression of Spy AcrIIA3 is toxic in E. coli. In the presence of IPIG (CRISPRi induction) and arabinose (AcrIIA3 induction), Spy AcrIIA3 is toxic in the presence or absence of sgRNA, indicating that its toxicity is independent of CRISPRi activity.

FIG. 12. Vector Map file for pPL2oexL, Related to FIG. 2 and FIG. 3. Genes and phage fragments to be tested for CRISPR-Cas9 inhibition in L. monocytogenes 10403s were cloned into pPL2oexL between pilyper and the FLAG tag. Native stop codons were included in pPL2oexL derivatives.

FIG. 13: acrIIA1 is very widespread across Firmicutes homologs are likely to inhibit Cas9 function in the organisms in which they are found. To identify new homologs of acrIIA genes with anti-CRISPR function, distinct members from the phylogenetic trees shown here were tested for anti-CRISPR activity in cell based assays. These homologs of known anti-CRISPR genes are being tested in a foreign bacterial system (Pseudomonas aeruginosa) to identify those with direct activity against SpyCas9.

FIG. 14: Phage plaque assays showing ten-fold dilutions of a control phage (D3) or a phage targeted (JBD30) by SpyCas9 with a JBD30-specific sgRNA in a heterologous host (Pseudomonas aeruginosa). Expression of the indicated anti-CRISPR acrIIA4 (positive control) or acrIIA1 inactivates SpyCas9.

FIG. 15A-B: 15A) A multi-sequence alignment of acrIIA2 homologs found in different Listeria mobile elements. (AcrIIA2a.1 (SEQ ID NO:69), AcrIIA2a.2 (SEQ ID NO:84), AcrIIA2b.1 (SEQ ID NO:108); AcrIIA2b,3 (SEQ II) NO:209); AcrIIA2c.1 (SEQ ID NO:97): and AcrIIA2c.2 (SEQ ID NO:98)). Tested homologs are indicated with colored arrows and a summary of the results are shown below the alignment, 15B) A table summarizing the sequence identity (at the amino acid level) between the different homologs and their accession numbers.

FIG. 16: Bacteriophage plaque assays with ten-fold serial dilutions phage JBD30 spotted on top of a lawn of P. aeruginosa expressing SpyCas9 and a sgRNA targeting phage JBD30. Phages will plaque in the absence of CRISPR activity. Cas9 and the sgRNA are induced with increasing amounts of arabinose from left to right (0.001%, 0.01%, 0.1%). In the absence of Cas9 (Acas9), the phages plaque fully, but in the present of Cas9 but no anti-CRISPR (vector), plaquing is reduced as Cas9 is induced. The provision of acriiA4 (positive control) fully blocks Cas9 at all levels. Only acrIIA2b.3 is comparable to acrIIA4 for its activity. The original acrIIA2a.1 is only partially active, with a slight improvement seen with acrIIA2b.1.

FIG. 17: Homologs of acrIIA3b found in Streptococcus species were tested for anti-CRISPR activity in heterologous system (P. aeruginosa) expressing SpyCas9 and an sgRNA. A summary of the results (data in FIG. 6) are shown in the table, indicating the species of origin for the anti-CRISPR, the sequence identity of Cas9 in that species to S. pyogenes, and similar information for the anti-CRISPR. Given the previously observed toxicity of acrIIA3a and acrIIA3b, these new proteins were assessed for toxicitiy (toxic?) and anti-CRISPR function (acr?).

FIG. 18: Spot titration of bacterial cells on LB agar plates. SpyCas9 was programmed with an sgRNA targeting the P. aeruginosa genome, thus killing the cell. Cells only survive if the anti-CRISPR is functional. Plates are showing 10-fold serial dilutions of cells plated on non-inducing (left column), ACR inducing only (middle column, to test ACR toxicity), or Cas9/sgRNA/ACR inducing plates (right column, to test ACR function). Genome being cleaved by Cas9 leads to death, unless anti-CRIS:PR blocks Cas9 function. Colonies on right-most panels indicate ACR activity.

FIG. 19: Summary of some of the data from Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Several polypeptide inhibitors (“Cas9-inhibiting polypeptides”) of Cas9 nuclease have been identified from phage. The Cas9-inhibiting polypeptides initially discovered from phage were designated AcrIIA1, AcrIIA2, AcrIIA3, and AcrIIA4.

The Cas9-inhibiting polypeptides described herein can be used in many aspects to inhibit unwanted Cas9 activity. For example, one or more Cas9-inhibiting polypeptide can be used to regulate Cas9 in genome editing, thereby allowing for some Cas9 activity prior to introduction of the Cas9-inhibiting polypeptide. This can be helpful, for example, in limiting off-target effects of Cas9. This and other uses are described in more detail below.

As set forth in the examples and sequence listing, a large number of Cas9-inhibiting polypeptides have been discovered. Examples of exemplary Cas9-inhibiting polypeptides include proteins comprising any of SEQ ID NOs: 1-169, or substantially (e.g., at least 50, 60, 70, 75, 80, 85, 90, 95, or 98%) identical amino acid sequences. In some embodiments, the polypeptides, in addition to having one of the above-listed sequences, will include other amino acid sequences or other chemical moieties (e.g., detectable labels) at the amino terminus, carboxyl terminus, or both. Additional amino acid sequences can include, but are not limited to tags, detectable markers, or nuclear localization signal sequences.

As noted in the examples, a number of the Cas9-inhibiting polypeptides have been shown to inhibit L. monocytogenes Cas9 as well as S. pyogenes (Spy) Cas9. It is believed and expected that the Cas9-inhibiting polypeptides described herein will also similarly inhibit other block II-A Cas9 proteins. As used herein, a “Cas9-inhibiting polypeptide” is a protein that inhibits function of the Cas9 enzyme in L. monocytogenes during a transformation efficiency assay. When a plasmid bearing a targeted DNA sequence and protospacer adjacent motif (PAM) is used to transform a strain with intact Cas9 function, the transformation event is prevented by Cas9, generating miniscule colonies under selection. This is compared to a plasmid with a non-targeted DNA sequence, which produces normal sized colonies when used to transform L. monocytogenes. The expression of a Cas9 inhibitor neutralizes Cas9 activity and leads to transformed, normal sized colonies of both the targeted and non-targeted plasmid. While it is believed the Cas9-inhibiting polypeptides' inhibitory activity can be measured in other ways, the above assay, presented in more detail in the Examples, is the assay for determining whether the Cas9-inhibiting polypeptide have activity.

The Cas9-inhibiting polypeptides can be introduced into any cell to inhibit Cas9 in that cell. In some embodiments, the cell contains Cas9 protein when the Cas9-inhibiting polypeptide is introduced into the cell. In other embodiments, the Cas9-inhibiting polypeptide is introduced into the cell and then Cas9 polypeptide is introduced into the cell.

Introduction of the Cas9-inhibiting polypeptides into the cell can take different forms. For example, in some embodiments, the Cas9-inhibiting polypeptides themselves are introduced into the cells. Any method for introduction of polypeptides into cells can be used. For example, in some embodiments, electroporation, or liposomal or nanoparticle delivery to the cells can be employed. In other embodiments, a polynucleotide encoding a Cas9-inhibiting polypeptide is introduced into the cell and the Cas9-inhibiting polypeptide is subsequently expressed in the cell. In some embodiments, the polynucleotide is an RNA. In some embodiments, the polynucleotide is a DNA.

In some embodiments, the Cas9-inhibiting polypeptide is expressed in the cell from RNA encoded by an expression cassette, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, the promoter is heterologous to the polynucleotide encoding the Cas9-inhibiting polypeptide. Selection of the promoter will depend on the cell in which it is to be expressed and the desired expression pattern. In sonic embodiments, promoters are inducible or repressible, such that expression of a nucleic acid operably linked to the promoter can be expressed under selected conditions. In some examples, a promoter is an inducible promoter, such that expression of a nucleic acid operably linked to the promoter is activated or increased.

An inducible promoter may be activated by presence or absence of a particular molecule, for example, doxycycline, tetracycline, metal ions, alcohol, or steroid compounds. In some embodiments, an inducible promoter is a promoter that is activated by environmental conditions, for example, light or temperature. In further examples, the promoter is a repressible promoter such that expression of a nucleic acid operably linked to the promoter can be reduced to low or undetectable levels, or eliminated. A repressible promoter may be repressed by direct binding of a repressor molecule (such as binding of the trp repressor to the trip operator in the presence of tryptophan). In a particular example, a repressible promoter is a tetracycline repressible promoter. In other examples, a repressible promoter is a promoter that is repressible by environmental conditions, such as hypoxia or exposure to metal ions.

In some embodiments, the polynucleotide encoding the Cas9-inhibiting polypeptide (e.g., as part of an expression cassette) is delivered to the cell by a vector. For example, in some embodiments, the vector is a viral vector. Exemplary viral vectors can include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors.

in some embodiments, the Cas9-inhibiting polypeptide or a polynucleotide encoding the Cas9-inhibiting polypeptide is delivered as part of or within a cell delivery system. Various delivery systems are known and can be used to administer a composition of the present disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, or receptor-mediated delivery.

Exemplary liposomal delivery methodologies are described in Metselaar et al., Mini Rev. Med. Chem. 2(4):319-29 (2002); O'Haggen et al., Expert Rev. Vaccines 2(2):269-83 (2003); O'Hagan, Curr. Drug Targets Infjct. Disord. 1(3):273-86 (2001); Zho et al., Biosci Rep. 22(2):355-69 (2002); Chikh et al., Biosci Rep. 22(2):339-53 (2002); Bungener et al., Biosci. Rep. 22(2):323-38 (2002); Park, Biosci Rep. 22(2):267-81 (2002); Ulrich, Biosci. Rep. 22(2):129-50; Lofthouse, Adv. Drug Deliv. Rev. 54(6):863-70 (2002); Zhou et al., J. Inmunmunother. 25(4):289-303 (2002); Singh et al., Pharm Res. 19(6):715-2.8 (2002); Wong et al., Curr. Med. Chem. 8(9):1123-36 (2001); and Zhou et al., Immunonmethods (3):229-35 (1994).

Exemplary nanoparticle delivery methodologies, including gold, iron oxide, titanium, hydrogel, and calcium phosphate nanoparticle delivery methodologies, are described in Wagner and Bhaduri, Tissue Engineering 18(1): 1-14 (2012) (describing inorganic nanoparticles); Ding et al., Mol Ther e-pub (2014) (describing gold nanoparticles); Zhang et al., Langmuir 30(3):839-45 (2014) (describing titanium dioxide nanoparticles); Xie et al., Curr Pharm Biotechnol 14(10):918-25 (2014) (describing biodegradable calcium phosphate nanoparticles); and Sizovs et al., J Am Chem Soc 136(1):234-40 (2014).

Introduction of a Cas9-inhibiting polypeptide as described herein into a prokaryotic cell can be achieved by any method used to introduce protein or nuclei acids into a prokaryote. In some embodiments, the Cas9-inhibiting polypeptide is delivered to the prokaryotic cell by a delivery vector (e.g., a bacteriophage) that deliver a polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, inhibiting Cas9 in the prokaryote could either help that phage kill the bacterium or help other phages kill it.

A Cas9-inhibiting polypeptide as described herein can be introduced into any cell that contains, expresses, or is expected to express, Cas9. Exemplary cells can be prokaryotic or eukaryotic cells. Exemplary prokaryotic cells can include but are not limited to, those used for biotechnological purposes, the production of desired metabolites, E. coli and human pathogens. Examples of such prokaryotic cells can include, for example, Escherichia coli, Pseudomonas sp., Corynebacterium sp., Bacillus subtitis, Streptococcus pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, Campylobacter jejuni, Francisella novicida, Corynebacterium diphtheria, Enterococcus sp., Listeria monocytogenes, Mycoplasma gallisepticum, Streptococcus sp., or Treponema denticola. Exemplary eukaryotic cells can include, for example, animal (e.g., mammalian) or plant cells. Exemplary mammalian cells include but are not limited to human, non-human primates, mouse, and rat cells. Cells can be cultured cells or primary cells. Exemplary cell types can include, but are not limited to, induced pluripotent cells, stem cells or progenitor cells, and blood cells, including but not limited to T-cells or B-cells.

In some embodiments, the cells are removed from an animal (e.g., a human, optionally in need of genetic repair), then Cas9, and optionally guide RNAs, for gene editing are introduced into the cell ex vivo, and a Cas9-inhibiting polypeptide is introduced into the cell. In some embodiments, the cell(s) is subsequently introduced into the same animal (autologous) or different animal (allogeneic).

In any of the embodiments described herein, a Cas9 polypeptide can be introduced into a cell to allow for Cas9 DNA binding and/or cleaving (and optionally editing), followed by introduction of a Cas9-inhibiting polypeptides as described herein. This timing of the presence of active Cas9 in the cell can thus be controlled by subsequently supplying Cas9-inhibiting polypeptides to the cell, thereby inactivating Cas9. This can be useful, for example, to reduce Cas9 “off-target” effects such that non-targeted chromosomal sequences are bound or altered. By limiting Cas9 activity to a limited “burst” that is ended upon introduction of the Cas9-inhibiting polypeptide, one can limit off-target effects. In some embodiments, the Cas9 polypeptide and the Cas9-inhibiting polypeptide are expressed from different inducible promoters, regulated by different inducers. These embodiments allow for first initiating expression of the Cas9 polypeptide followed later by induction of the Cas9-inhibiting polypeptide, optionally while removing the inducer of Cas9 expression.

In some embodiments, a Cas9-inhibiting polypeptide as described herein can be introduced (e.g., administered) to an animal (e.g., a human) or plant. This can be used to control in vivo Cas9 activity, for example in situations in which CRISPR/Cas9 gene editing was performed in vivo, or in circumstances in which an individual is exposed to unwanted Cas9, for example where a bioweapon comprising Cas9 is released.

In some embodiments, the Cas9-inhibiting polypeptides or a polynucleotide encoding the Cas9-inhibiting polypeptide, in administered as a pharmaceutical composition. In some embodiments, the composition comprises a delivery system such as a liposome, nanoparticle or other delivery vehicle as described herein or otherwise known, comprising the Cas9-inhibiting polypeptides or a polynucleotide encoding the Cas9-inhibiting polypeptide. The compositions can be administered directly to a mammal (e.g., human) to inhibit Cas9 using any route known in the art, including e.g., by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, or intrademal), inhalation, transdermal application, rectal administration, or oral administration.

The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

EXAMPLES Example 1

Results

CRISPR-Cas9 in Listeria monocytogenes Targets Foreign DNA

Listeria monocytogenes is a facultative intracellular food-borne pathogen with a well characterized phage population. Many L. monocytogenes isolates have type II-A CRISPR-Cas systems (Sesto, N. et al. (2014). PLoS Genet, 10, e1004065) and their CRISPR spacers possess identity to many virulent, temperate, and integrated phages (Di, H. et al, (2014) Biochem Biophys Res Commun., 454, 399-403; Sesto, N. et al. (2014). PLoS Genet, 10, e1004065). However, there is no experimental evidence of canonical CRISPR-Cas function. We analyzed 275 genomes of L. monocytogenes and identified typeII-A CRISPR-Cas9 systems (Lmo Cas9) in 15% (n=41) of genomes (FIG. 1B). Interestingly, we found eight genomes (3% of the total), with examples of self-targeting (FIG. 1B and 1C) although CRISPR-cas9 is anticipated to be functional as the CRISPR repeat, PAM, tracrRNA, Cas9, and the associated promoters lack obvious inactivating mutations (FIG. 6A-E), We predicted that these strains possess inhibitors of CRISPR-Cas9 that allow the stable co-existence of a spacer-protospacer pair.

To test whether inhibitors were encoded by the prophages of L. monocytogenes, we first established the functionality of uninhibited CRISPR-Cas9 in an L. monocytogenes strain (10403s) that does not exhibit self-targeting. To test the activity of this system we designed a plasmid (pT) possessing a targeted protospacer (i.e. a sequence that is complementary to a natural spacer in the CRISPR array) along with a cognate protospacer adjacent motif (PAM), a three base motif that is necessary for Cas9 binding (FIG. 2A). We measured the transformation efficiency of 10403s with either pT or a control plasmid possessing a non-targeted sequence with an identical plasmid backbone (pNT). To simplify downstream analysis, a prophage cured version of 10403s ( cure) was used for all downstream experiments since it was indistinguishable from wt10403s in this assay (FIG. 7). Transformation with pT yielded miniscule colonies relative to pNT (FIG. 2B, leftmost panel), although the number of colonies that emerged upon prolonged incubation were the same (FIG. 2C, see Discussion for further analysis). To confirm that this phenotype was the result of CRISPR-Cas9 interference, we constructed a cas9-deletion strain. Transformation of this strain with pT and pNT produced colonies of indistinguishable size (FIG. 2B 2^(nd) left panel). However, adding back cas9 to the L. monocytogenes chromosome under a constitutively active promoter completely inhibited transformation with pT (FIG. 2C). Together, these experiments demonstrate that Cas9 is functional in the L. monocytogenes strain 10403s at both endogenous and overexpressed levels, and limits transformation with a plasmid bearing a protospacer.

Resident Prophages Inactivate CRISPR-Cas9 in L. monocytogenes

To determine whether CRISPR-Cas9 may be disabled in a strain with self-targeting spacers, we examined immunity function in L. monocytogenes strain J0161, whose spacer 16 perfectly matches a prophage (φJ0161a) in the same genome (FIG. 1C). We could not detect any clearly deleterious CRISPR-Cas mutations in J0161, suggesting that this self-targeting scenario was the result of inhibition and not loss of function (FIG. 6B-F). Since the type II-A CRISPR array of J0161 is distinct from that of 10403s, a J0161-specific targeted plasmid (pT_(J0161)) was used to test the function CRISPR-Cas9 in J0161. Consistent with the inactivation implied by self-targeting, there was no significant difference in transformation efficiency or colony size to distinguish pT_(J0161) from pNT (FIGS. 2B and 2C, rightmost panel). Thus, we reasoned that that the J0161 genome may encode Cas9 inhibitors.

In search of the genetic basis for CRISPR-Cas9 inactivation in J0161, we focused on the prophage φJ0161a as a likely source of an inhibitor gene because it contained the self-targeted sequence in this strain. To determine whether φJ0161a contained an inhibitor, the prophage-cured strain of 10403s was lysogenized with φJ0161a and assayed for CRISPR-Cas9 functionality by plasmid transformation. While φcure10403s strain targeted pT, the acquisition of φJ0161a was sufficient to inactivate CRISPR-Cas9 function (FIGS. 2B, 3^(rd) panel from left and 2C, 4^(th) from left), suggesting that this prophage encodes an inhibitor of CRISPR-Cas9. The φJ0161a prophage also inactivated plasmid targeting in a strain constitutively expressing cas9, suggesting that the inhibitory mechanism does not operate by disrupting natural regulation of the cas9 promoter (FIG. 2C. 5^(th) from left).

Given that φJ0161a inhibited CRISPR-Cas9 function, and the endogenous prophage φ10403s did not, we compared the genomes of these two closely related phages to identify the regions of difference (FIG. 2D). In addition to sharing 39 core phage genes with >40% protein sequence identity, ten non-overlapping unique dusters of genes were identified (cluster boundaries were chosen based on predicted operon structure, with 1-12 genes per cluster). Each cluster was cloned and integrated into the genome of prophage-cured 10403s and assayed for CRISPR-Cas9 function. Of the ten fragments, seven were successfully introduced into L. monocytogenes, while three fragments could not be inserted in the L. monocytogenes genome and were presumably toxic in isolation. Plasmid transformation assays revealed that φJ0161a fragment 1 was the only fragment capable of inhibiting CRISPR-Cas9, indicating that his fragment encoded at least one CRISPR-Cas9 inhibitor (FIG. 2D, FIG. 8). Expressing the individual genes from this four-gene fragment led to the conclusive identification of two anti-CRISPR genes, LMO_03146 and LMOG_03147 (herein referred to as acrIIA1 and acrIIA2, respectively; FIGS. 2B and 2D). Deletion of both acrIIA1 and acrIIA2 from a 10403s::φJ0161a lysogen restored CRISPR-Cas9 function, confirming that these are the only anti-CRISPR genes in φJ0161a (FIG. 2B).

Anti-CRISPR Loci are Widespread in L. monocrytogenes

To identify additional type II-A anti-CRISPRs, the genomic position analogous to that of acrIIA1 and acrIIA2 in related L. monocytogenes prophages was examined. A recurring anti-CRISPR (acr) locus containing acrIIA1 within in a small operon (2-5 genes) of highly conserved gene order was identified between the left integration site and the genes involved in cell lysis (FIG. 3A). We identified five novel protein families conserved within acr loci. To test these, we cloned and integrated representatives into the 10403s genome and assayed the transformation efficiency of pT and pNT. Two new genes were identified that were capable of CRISPR inactivation (acrIIA³ and acrIIA4), while the remaining three (orfC, D, E) were not (FIG. 3A, FIG. 9).

To determine whether CRISPR-Cas9 inactivation in L. monocytogenes is pervasive, we next analyzed the conservation pattern for each anti-CRISPR. Although each acrIIA gene was sufficient to inactivate CRISPR-Cas9 in isolation, we observed a common presence of acrIIA1 in most acr loci. Nearly all instances (91%) of acrIIA2-4 were found upstream of the helix-turn-helix (HTH) motif-containing acrIIA1, suggesting that this gene may be a marker for acr loci (FIG. 3B). The most common scenario we observed in 119 acr loci were either acIIA1-2 or acrIIA1-2-3, representing 66% of acr loci. In total, acrIIA genes were identified in 25% of L. monocytogenes genomes, with approximately 50% oft monocytogenes cas9-containing strains possessing at least one anti-CRISPR in the same genome (FIG. 3C). Many instances of L. monocytogenes genomes possessing multiple acrIIA-encoding prophages were also identified (Supplementary Table 1). Furthermore, at least one acrIIA gene was found in the genomes of all eight instances of self-targeting that were initially identified (FIG. 1B, Supplementary Table 1), explaining how these scenarios are stable. Together, these data suggest widespread prophage-mediated inactivation of CRISPR-Cas9 in L. monocytogenes.

TABLE S1 acrIIA Gene Conservation, Related to FIGS. 1B 4B and 4C genomes that lack cas9 and all known acrIIA genes 264 genomes that contain cas9 but lack all known acrIIA genes 33 genomes contain cas9 and acrIIA genes 37 genomes that lack cas9, but contain acrIIA genes 65 unpaired acr genes control (cysS) acrIIA3 acrIIA2 acrIIA1 acrIIA4 Cas9 ST strains AEO05249.1 AEO07576.1 AKI48207.1 AKI50529.1 AMD50972.1 AMD53187.1 AMR52783.1 AMR55031.1 EEW14429.1 EEW14776.1 KES84268.1 KES86621.1 KET22766.1 KET25022.1 KET54818.1 KET56390.1 KEU03893.1 KEU00315.1 KEU81061.1 KEU82991.1 KEU92860.1 KEU94779.1 KEV01501.1 KEV04018.1 KEV13316.1 KEV15767.1 KEV34890.1 KEV37443.1 KEV87445.1 KEV88657.1 KEW12308.1 KEW13456.1 KEW54858.1 KEW57091.1 KEX56334.1 KEX57585.1 KEX62909.1 KEX64108.1 KEX85751.1 KEX84898.1 KFL18307.1 KFL20187.1 KHK15389.1 KHK15281.1 KPV81115.1 KPV80639.1 KTA33634.1 KTA27954.1 KTA40577.1 KTA36142.1 KTA59125.1 KTA59526.1 KTE88758.1 KTE86646.1 KXS69410.1 KXS73069.1 KXX03397.1 KXX04680.1 KXX27153.1 KXX27120.1 KYH47795.1 KYH48716.1 KEX06234.1 KEX03765.1 AGR07328.1 AGR09779.1 EFG02722.1 EFG02166.1 EFG02165.1 FG02164.1 EFG02048.1 FSL J1-194 AEO02308.1 AEO04363.1 AEO04364.1 AEO04689.1 AEO04756.1 J0161 AEO04690.1 AKI51021.1 AKI52063.1 AKI52062.1 AKI52061.1 AKI53425.1 L1846 AKI53105.1 AKI53106.1 AKI53107.1 AKI41163.1 AKI40129.1 AKI40130.1 AKI40131.1 AKI42028.1 L2626 AGR26778.1 AGR27459.1 AGR27460.1 AGR27297.1 AGR27343.1 R2-502 AGR27461.1 EEW19474.1 EEW20426.1 EEW20201.1 R2-503 AHJ04747.1 AHJ02948.1 AHJ02947.1 AHJ02946.1 AHJ04249.1 WSLC1001 AKI42516.1 AKI43551.1 AKI43550.1 AKI43549.1 AKI44910.1 EXL23533.1 EXL25968.1 EXL23613.1 EZH70416.1 EZH69742.1 EZH71062.1 EZH69029.1 EZH69562.1 EZH71063.1 KEU59490.1 KEU52815.1 KEU52814.1 KEU52813.1 KEU61049.1 KHK05112.1 KHK09045.1 KHK09044.1 KHK04755.1 KHK11936.1 KHK09043.1 KID24070.1 KID23649.1 KID23650.1 KID23651.1 KID22034.1 KID25721.1 KID25720.1 KKB89786.1 KKB89545.1 KKB87491.1 KKB87492.1 KKB87210.1 KKB89544.1 KKB89543.1 KTA33603.1 KTA31236.1 KTA31235.1 KTA28092.1 KTA28352.1 KTA31234.1 KTA58546.1 KTA51620.1 KTA51621.1 KTA51622.1 KTA50572.1 KXS57839.1 KXS58608.1 KXS58607.1 KXS58606.1 KXS56938.1 KXS59964.1 EAL04996.1 EAL06504.1 EAL05810.1 EAL05809.1 EAL05449.1 EAL06505.1 EEW23167.1 EEW22373.1 EEW22374.1 EEW23439.1 EEW22432.1 EEW23440.1 EFF99811.1 EFG00297.1 EFG00298.1 EFG00182.1 EFF99171.1 EFG00183.1 EHY63973.1 EHY61390.1 EHY61427.1 EXL17326.1 EXL17712.1 EXL17711.1 EXL16255.1 KTA40536.1 KTA33666.1 KTA31190.1 KTA31189.1 KTA29552.1 KTA33667.1 KXS59387.1 KXS56902.1 KXS64534.1 KXS57719.1 KXS62773.1 KXW90032.1 KXW85500.1 KXW85495.1 KXW85912.1 KXW90865.1 KXW85497.1 KTA67982.1 KTA68177.1 KTA68618.1 AGR14764.1 AGR15693.1 AGR15756.1 ALU77418.1 ALU78083.1 ALU77910.1 EFK41798.1 EFK41083.1 EFK42981.1 KHK20071.1 KHK19909.1 KHK21360.1 KHK20265.1 KHK17523.1 KHK22389.1 KHK26884.1 KHK28212.1 KHK28213.1 KHK29000.1 KHK32359.1 KHK33774.1 KHK33773.1 KHK34506.1 KID12814.1 KID20146.1 KID20145.1 KID19562.1 KID16736.1 KID21567.1 KID21568.1 KID14794.1 KID25109.1 KID27661.1 KID27662.1 KID22855.1 KXX34587.1 KXX34834.1 KXX34335.1 KXX35452.1 KXX34219.1 ACK40737.1 ACK39691.1 ACK39692.1 ACK39693.1 ACK40885.1 AEH91268.1 AEH92315.1 AEH92314.1 AEH92313.1 AEH91120.1 ALU81417.1 ALU80614.1 ALU80615.1 ALU80616.1 EXL28212.1 EXL28247.1 EXL28248.1 EXL28249.1 KES32042.1 KES29691.1 KES29690.1 KES29689.1 KES38767.1 KES36191.1 KES36190.1 KES36189.1 KES64642.1 KES69056.1 KES69057.1 KES69058.1 KET22488.1 KET20225.1 KET20226.1 KET20227.1 KET33547.1 KET33008.1 KET33009.1 KET33010.1 KET65150.1 KET67219.1 KET67220.1 KET67221.1 KEU38314.1 KEU32638.1 KEU32639.1 KEU32640.1 KEU70290.1 KEU73964.1 KEU69222.1 KEU69221.1 KEU77521.1 KEU79801.1 KEU73965.1 KEU73966.1 KEU79802.1 KEU79803.1 KEU85819.1 KEU87627.1 KEU87628.1 KEU87629.1 KEW39277.1 KEW37877.1 KEW37876.1 KEW37875.1 KEW47138.1 KEW38798.1 KEW38797.1 KEW38796.1 KEW52188.1 KEW54596.1 KEW54597.1 KEW54598.1 KEW87000.1 KEW91381.1 KEW91380.1 KEW91379.1 KEX05231.1 KEX03851.1 KEX03850.1 KEX03849.1 KEX16623.1 KEX13878.1 KEX13879.1 KEX13880.1 KEX48257.1 KEX45733.1 KEX45732.1 KEX45731.1 KEX44142.1 KEX49273.1 KEX49272.1 KEX49271.1 KHK37385.1 KHK39424.1 KHK39423.1 KHK39422.1 KLI10624.1 KLI10452.1 KLI10451.1 KLI10195.1 KLI10194.1 KLI12476.1 KLI12475.1 KLI10251.1 KNX95479.1 KNX95907.1 KNX95906.1 KNX94640.1 KNX94641.1 KPJ28401.1 KPJ30389.1 KPJ30390.1 KPJ30391.1 KPV83306.1 KPV85471.1 KPV85472.1 KPV85473.1 KTA46249.1 KTA44520.1 KTA44521.1 KTA44522.1 KTA45326.1 KTA51238.1 KTA50253.1 KTA50252.1 KTA50251.1 KTA50988.1 KTA64947.1 KTA62142.1 KTA62143.1 KTA62144.1 KXS86581.1 KXS85159.1 KXS85158.1 KXS85157.1 KXX46503.1 KXX46300.1 KXX46299.1 KXX46298.1 KXX49128.1 KXX48607.1 KXX48606.1 KXX48605.1 KXX50214.1 KXX49264.1 KXX49263.1 KXX49262.1 AKI55317.1 AKI56173.1 AKI56172.1 AMD23307.1 AMD24317.1 AMD24318.1 KKD52037.1 KKD49091.1 KKD49092.1 KTA47721.1 KTA46387.1 KTA46388.1 KTA50332.1 KTA52328.1 KTA52327.1 KXS77556.1 KXS77365.1 KXS79013.1 KXS78354.1 KXX11384.1 KXX11218.1 KXX17306.1 KXX17138.1 KXX19133.1 KXX18287.1 KES91745.1 KES96882.1 KES96881.1 KET71424.1 KET73263.1 KET73262.1 KET90504.1 KET94691.1 KET94692.1 KEV66815.1 KEV69928.1 KEV69929.1 KEV93156.1 KEV93282.1 KEV93281.1 KEW04107.1 KEW08181.1 KEW08182.1 KEW04863.1 KEW09554.1 KEW09555.1 KEW11516.1 KEW17021.1 KEW17020.1 KEW59026.1 KEW65182.1 KEW65181.1 KEX05590.1 KEX05985.1 KEX05984.1 KHK13841.1 KHK12400.1 KJJ91084.1 KJJ91611.1 KJJ91612.1 KJQ95289.1 KJQ94313.1 KJQ94314.1 KJQ98292.1 KJQ95811.1 KJQ95812.1 KJR57725.1 KJR51141.1 KJR51140.1 KJR58524.1 KJR60208.1 KJR60209.1 KKD51859.1 KKD43688.1 KTA41666.1 KTA35071.1 KTA65269.1 KTA63900.1 KXF69083.1 KXF66382.1 KXF66381.1 AGR12413.1 AGR07062.1 AGR07061.1 AAT03038.1 ADB67037.1 ADB70126.1 AEO24539.1 AEO37792.1 AFH78832.1 AGR02736.1 AGR04913.1 AGR16038.1 AGR21403.1 AGR21930.1 AGR32499.1 AHF28095.1 AHF30972.1 AHF33963.1 AHF36954.1 AHF39945.1 AHF42886.1 AHI68925.1 AHJ37147.1 AHN31516.1 AHY99532.1 AIL67941.1 AIZ37538.1 AJA81957.1 AJT44045.1 AKG84402.1 AKG87228.1 AKI45409.1 AKP37411.1 AKS52855.1 ALQ13660.1 ALQ15375.1 ALQ19576.1 ALQ21479.1 ALQ25193.1 ALU83475.1 ALX67727.1 AMD26187.1 ANE38117.1 EAL07955.1 EFD91450.1 EFF96046.1 EFR95169.1 EGF38890.1 EGJ23745.1 ERH76184.1 ERH76295.1 ERH77379.1 ERH83604.1 ERH85425.1 EUJ16754.1 EXL13590.1 EXL14999.1 EXL25668.1 KEK05609.1 KEK07032.1 KES28051.1 KES28301.1 KES38071.1 KES42071.1 KES42797.1 KES48493.1 KES50725.1 KES54322.1 KES54965.1 KES55696.1 KES63074.1 KES63309.1 KES72038.1 KES79358.1 KES79767.1 KES80806.1 KES82973.1 KES86788.1 KES92539.1 KET00546.1 KET04697.1 KET06207.1 KET08149.1 KET08880.1 KET13375.1 KET15678.1 KET17220.1 KET30859.1 KET35076.1 KET38677.1 KET39554.1 KET41316.1 KET44817.1 KET47850.1 KET53482.1 KET55834.1 KET60044.1 KET62217.1 KET69318.1 KET74754.1 KET76626.1 KET81048.1 KET85737.1 KET86860.1 KET91339.1 KET98444.1 KEU01781.1 KEU03644.1 KEU06655.1 KEU10723.1 KEU12483.1 KEU18590.1 KEU21146.1 KEU23067.1 KEU24780.1 KEU30341.1 KEU36177.1 KEU37301.1 KEU43040.1 KEU43555.1 KEU46678.1 KEU51513.1 KEU55425.1 KEU55498.1 KEU58686.1 KEU64129.1 KEU68275.1 KEU69241.1 KEU77548.1 KEU86942.1 KEU90182.1 KEV00307.1 KEV00562.1 KEV06907.1 KEV11638.1 KEV12282.1 KEV14240.1 KEV21793.1 KEV22298.1 KEV26044.1 KEV32026.1 KEV32063.1 KEV35626.1 KEV42967.1 KEV44621.1 KEV49515.1 KEV49947.1 KEV52716.1 KEV53451.1 KEV60716.1 KEV63470.1 KEV71076.1 KEV74160.1 KEV76378.1 KEV76776.1 KEV79179.1 KEV81224.1 KEV88927.1 KEV93759.1 KEV95283.1 KEW03640.1 KEW09573.1 KEW18769.1 KEW19179.1 KEW21604.1 KEW27010.1 KEW31226.1 KEW33386.1 KEW36305.1 KEW42387.1 KEW44715.1 KEW57718.1 KEW65692.1 KEW66741.1 KEW71344.1 KEW71596.1 KEW72422.1 KEW77363.1 KEW79497.1 KEW80113.1 KEW84366.1 KEW93848.1 KEW95807.1 KEW96317.1 KEX09756.1 KEX11628.1 KEX19359.1 KEX20220.1 KEX23616.1 KEX29531.1 KEX29582.1 KEX33273.1 KEX39294.1 KEX42941.1 KEX46988.1 KEX50326.1 KEX51799.1 KEX64668.1 KEX68538.1 KEX69000.1 KEX72953.1 KEX73328.1 KEX77547.1 KEX82199.1 KFZ71375.1 KGJ75182.1 KGJ80867.1 KGR20308.1 KHK07430.1 KHK19170.1 KHK26142.1 KHK36612.1 KHQ60145.1 KHQ69332.1 KHS60750.1 KHS61164.1 KID14246.1 KJH24122.1 KJJ89955.1 KJQ79905.1 KJQ83378.1 KKF72182.1 KKO43304.1 KNX50880.1 KNX54024.1 KNX58068.1 KNX62105.1 KNX63287.1 KNX66597.1 KNX70553.1 KNX72130.1 KOX87096.1 KPJ27561.1 KQC81886.1 KQC82004.1 KRJ93270.1 KRW87784.1 KSZ43192.1 KSZ44687.1 KSZ45883.1 KSZ48351.1 KTA43969.1 KXF63597.1 KXS57277.1 KXS68541.1 KXS69730.1 KXS75040.1 KXS83236.1 KXW87734.1 KXW89246.1 KXW94597.1 KXW97284.1 KXX00507.1 KXX03707.1 KXX09006.1 KXX14888.1 KXX23313.1 KXX28906.1 KXX32727.1 KXX35528.1 KXX37642.1 KXX42799.1 KYB35881.1 KYB36139.1 KYB36717.1 KYB42077.1 KYB43947.1 SLCC5850 was no included in the conservation pattern analysis CBY50703.1 CBY51763.1 CBY51762.1 CBY51761.1 CBY53074.1 slcc5850

Previous HTH-containing anti-CRISPR associated (aca) genes were used as markers to identify novel type I anti-CRISPR genes (Pawluk, A. et al. (2016). Nature Microbiology, 1, 1-6), although the aca genes did not have anti-CRISPR activity themselves. We hypothesized that acrIIA1 could fulfill the role of such a marker. A comprehensive phylogenetic analysis of acrIIA1 was conducted, revealing homologs detected widely across Firmicutes, in both mobile elements and core genomes (FIG. 4A). A family of distantly related acrIIA1 homologs were identified in Listeria genomes, exemplified by orfD, an HTH-containing gene that had been independently identified as an acr locus member (FIG. 3A). While this gene lacked anti-CRISPR activity in a functional assay, its co-occurrence with acrIIA4 in plasmid pLMIV suggests that the broad acrIIA1/orfD superfamily could be used as a marker to identify new acr genes (FIG. 3A). Future work will be necessary to determine whether the HTH-containing genes in these systems serve as effective markers for novel anti-CRISPR discovery.

To determine the homology landscape of acrIIA2-4, similar phylogenetic analyses were performed. Unlike acrIIA1, which was widespread across Firmicutes core genomes, these other three acr genes were mostly restricted to prophages in Listeria. Three distinct sequence families of acrIIA2 were identified, found only within Listeria siphophages (a family of long tailed, non-contractile phages) (FIG. 4B), while two acrIIA3 families were observed in the genomes of siphophages Listeria and Streptococcus (FIG. 4C). Lastly, acrIIA4 was observed in two distinct sequence families, one in Listeria, siphophages and plasmids, and the other in a group of obligate virulent myophages (long contractile tailed phages) (FIG. 4D). While acrIIA2 and acrIIA3 were nearly always found with acrIIA1, acrIIA4 often occurred in the absence of acrIIA1 homologs in phages and mobile elements of Listeria. For example, the family of acrIIA4 in virulent phages are distinct in that they have an ˜70 amino acid C-terminal extension in the predicted protein and do not occur with the HTH-containing genes acrIIA1 or orfD, suggesting potential mechanistic and evolutionary distinctions between these acrIIA4 families. Together, these analyses reveal ample sequence space for surveying homologous acr genes for specificity determinants and suggest an active arms race between cas9 and mobile elements in L. monocytogenes.

AcrIIA2, AcrIIA3, and AcrIIA4 inhibit S. pyogenes Cas9

To determine the versatility of the Lmo Cas9 AcrIIA proteins, we asked whether these inhibitors were functional on the related Cas9 protein from S. pyogenes (Spy, 53% identical to Lmo Cas9). This ortholog has been used widely for biotechnological applications as an RNA-guided nuclease (Barrangou, R., and Doudna, J. A. (2016), Nature Biotechnology, 34, 933-941), as well as for programmable gene repression by a catalytically deactivated mutant (dCas9) for programmable gene repression (Gilbert, L. A. et al. (2013). Cell 154, 442-451; Qi, L. S. et al. (2013). Cell, 152, 1173-1183). Using an E. coli strain that carries Spy dCas9, we tested whether AcrIIA proteins block dCas9 from interfering with transcription of a chromosomal RFP reporter gene (FIG. 5A). In a genetic background lacking inhibitors, the presence of an sgRNA and dCas9 reduced RFP fluorescence to 2.6% relative to a strain with no guide RNA. acrIIA2 partially blocked dCas9 function with fluorescence rising to 25%, while acrIIA4 nearly completely blocked dCas9, with fluorescence at 85% of the no guide control (FIG. 5B). We could not obtain meaningful data from acrIIA3 because the protein was toxic to E. coli. This lowered the recorded cell count during flow cytometry (see FIG. 10a ) and lead to large variability in the fluorescence measurements. A homolog of AcrIIA3 from S. pyogenes (accession number: AND04610.1) with 45% sequence identity to Lmo_acrIIA3 was also tested, but resulted in impaired growth of E. coli (FIG. 10b ). The mechanism of acrIIA3 toxicity remains to be determined. We conclude the acrIIA2 and acrIIA4 inhibit Spy dCas9 in E. coil to different degrees.

Given the common application of Spy Cas9 in eukaryotic cells, we next tested the AcrIIA proteins for their ability to block gene editing in human cells. HEK293T cells with an inducible eGFP reporter gene were transiently transfected with a plasmid expressing both Spy Cas9 and an sgRNA targeting eGFP in the presence or absence of vectors expressing human codon optimized AcrIIA proteins. After allowing gene editing to proceed for 36 h, eGFP was induced for 12 h, and cellular fluorescence was then measured by flow cytometry (FIG. 5C). In the presence of Cas9 and the eGFP sgRNA, gene editing resulted in a 25% decrease in the number of GFP positive cells, while co-expression with acrIIA2 or acrIIA4 prevented Cas9-based gene editing (FIG. 5D). We additionally tested the S. pyogenes homolog of acrIIA3 (Spy_acrIIA3) in this assay, which was not toxic in human cells, but had no impact on Cas9 function. acrIIA1 was non-functional in human cells, as was orfA, a negative control that has no anti-CRISPR activity in L. monocytogenes. Taken together with dCas9 experiments in E. coli, these data demonstrate the utility of the AcrIIA2 and AcrIIA4 proteins to inhibit the function of an orthologous Cas9 in heterologous hosts. These reagents, therefore, represent new tools in the CRISPR-Cas9 genome engineering toolkit.

Discussion

Phage-encoded inhibitors of bacterial immune systems emerge due to the strong selective pressures in the evolutionary arms race between these two entities (Samson, J. E. et al. (2013). Nat Rev Micro, 11, 675-687). The first identification of phage encoded anti-CRISPRs in type I CRISPR-Cas systems hinted that more CRISPR inhibitors existed, but methods were lacking for their discovery. Here, we present a bioinformatics strategy that uses “self-targeting” as a genomic marker for CRISPR-Cas inhibitor genes (FIG. 1A). This approach led to the identification of four different type II-A CRISPR-Cas9 inhibitors (FIG. 3A), which are collectively present in half of all Cas9-encoding L. monocytogenes genomes, including all genomes with self-targeting (FIG. 3C). We anticipate that this approach will be helpful for identifying acr genes in other CRISPR-Cas systems, although a distinct mechanism for tolerance of self-targeting has been described for type III systems (Goldberg, G. W. et al. (2014). Nature 514, 633-637; Samai, P. et al. (2015). Cell, 161, 1164-1174).

To facilitate the identification of AcrIIA proteins, we first demonstrate a functional CRISPR-Cas9 system in L. monocytogenes (FIG. 2B). Previous studies of CRISPR-Cas in this organism have focused on the type I-B system and an associated I-B derived CRISPR orphan array lacking cas genes (Mandin, P. et al. (2007). Nucleic Acids Research, 35, 962-974; Sesto, N. et al. (2014). PLoS Genet, 10, e1004065). Although no canonical CRISPR-Cas function had been established for either system previously, the orphan array was shown to be processed by a host ribonuclease to generate non coding RNAs (Mandin, P, et al. (2007). Nucleic Acids Research, 35, 962-974; Sesto, N. et al. (2014). PLoS Genet, 10, e1004065). To observe function for the II-A CRISPR-Cas system, we used a standard transformation efficiency assay, showing that Cas9 function in strain 10403s is sufficient to limit transformation of a plasmid in a sequence specific manner (FIG. 2A-C). Given the small colony phenotype observed during transformation of 10403s with pT, we suspect that endogenous levels of cas9 expression are not sufficient to totally clear the plasmid, leading to maintained plasmid in a tiny colony. Confirming this, increased expression of cas9 resulted in a complete elimination of transformants in this assay (FIG. 2C). Given that φJ0161a can inhibit this overexpressed CRISPR-Cas9 system (FIG. 2C), we conclude that the identified inhibitors are able to block both endogenous and overexpressed Cas9 function.

To identify candidate anti-CRISPR genes, related prophages from CRISPR-active strain 10403s to a prophage that inhibits CRISPR from strain J0161 were compared, and a process of elimination cloning approach was taken (FIG. 2D). The identification of two isolated acr genes was confirmed for acrIIA1 and acrIIA2 genes that are present in φJ0161. In searching for more anti-CRISPRs, we find that conserved genomic positioning in related. phages is a good proxy for identifying distinct type II-A Cas9 inhibitor proteins, despite a lack of sequence conservation between the proteins themselves (FIG. 3A). This has been observed previously in studies of Type I-F and I-E anti-CRISPRs (Bondy-Denomy et al. (2013). Nature, 493, 429-432; Pawluk, A. et al. (2014). mBio 5, e00896). In L. monocytogenes, the high prevalence of Cas9 inhibitors in prophages suggests the widespread inactivation of CRISPR-Cas9 function (FIG. 3C). At present, we do not understand whether there is a mechanistic link to explain the common co-occurrence of acIIA1 with other anti-CRISPRs (FIGS. 3A and 3B). Although this gene is sufficient to inactivate CRISPR-Cas9 function in a plasmid challenge assay, we speculate that it could act as a co-factor or regulator of other acrII4 genes during infection or lysogeny, thus explaining the genomic associations observed. Future work will be necessary to understand whether AcrIIA1 is, in fact, a bi-functional protein in this regard and more broadly, whether the superfamily is a marker for acr genes.

Phylogenetic analyses demonstrate common occurrences of acrIIA2-4 in mobile elements in Listeria mobile elements (FIG. 4). This likely facilitates horizontal gene transfer in this organism by blocking Cas9-based targeting and adaptation (Heler, R. et al. (2015) Nature 519, 199-202). In addition to the family of prophages where these acrIIA genes were first identified, homologs were also found in distant siphophages, myophages and plasmids. Most notably, the acrIIA4 homologs encoded by virulent myophages did not have acrIIA1 superfamily homologs in their vicinity. Furthermore, the presence of acrIIA1 and acrIIA3 homologs in genera outside of Listeria demonstrates that CRISPR-Cas9 inactivation may be common-place in the Firmicutes.

To inactivate CRISPR-Cas9 function, many mechanisms are possible, in theory. By demonstrating the efficacy of acrIIA2 and acrIIA4 in heterologous hosts with engineered elements (i.e. cas9 promoter, sgRNA design and promoter) we conclude that transcriptional repression is unlikely. Type I anti-CRISPRs function by binding directly to the Cas proteins required for interference and preventing DNA binding or DNA cleavage (Bondy-Denomy, J et al. (2015). Nature, 526, 136-139). By extension, we expect a similar mechanism for acrIIA2 and acrIIA4, given their ability to function in human cells. The enhanced efficacy of acrIIA2 in the cleavage-based Cas9 assay in human cells relative to the dCas9 based assay suggests that it may inhibit both binding and cleavage to some degree, with cleavage inhibition manifesting as a full inactivation of Cas9 function. In the case of acrIIA4, DNA-binding is clearly inhibited, although whether this is through a direct interaction with Cas9 remains to be seen.

The identification and future mechanistic dissection of type II-A inhibitors will provide valuable new reagents for studying canonical CRISPR-Cas9 function in natural and engineered settings. The ability of AcrIIA proteins to block Spy Cas9 in E. coil and human cells suggests that these proteins can provide a post-translational “off-switch” for Cas9. This could add a layer of regulation on this powerful system that can be applied in eukaryotic systems to control genome engineering. This new addition to the CRISPR-Cas9 toolbox could enable new applications, such as specifically reversing the effects of dCas9 binding to a genomic locus, or limiting the amount of time that Cas9 is active in the nucleus to reduce off-target gene editing. It will be important to expand the search for inhibitor proteins to continue to exploit the abundant tools provided to us from the phage-bacteria arms race.

REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Cell Lines HEK293T ATCC N/A Experimental Models: Organisms/Strains Listeria monocytogenes 10403s Laboratory ncbi.n1m.nih.gov/Taxonomy/ of Daniel Browser/wwwtax.cgi?mode= Portnoy Info&id=393133&1v1= 3&1in=f&keep=1& srchmode=1&unlock Listeria monocytogenes 10403s derivatives this paper see Table S2 Listeria monocytogenes J0161 Laboratory ncbi.n1m.nih.gov/Taxonomy/ of Martin Browser/wwwtax.cgi?id=393130 Wiedmann Listeria monocytogenes SLCC2482 Ariane ncbi.n1m.nih.gov/Taxonomy/ Pietzka Browser/wwwtax.cgi?id=863767 Lisieria mortoeytogenes SLCC2540 Ariane ncbi.n1m.nih.gov/Taxonomy/ Pietzka Browser/wwwtax.cgi?id=879089 Escherichia coli BW25113 derivatives this paper see Table S2 Recombinant DNA pBAD24 Laboratory ncbi.n1m.nih.gov/nuccore/ of Carol X81837.1 Gross pBAD24-derivative plasmids this paper see Table S2 pdCas9-bacteria Addgene addgene.org/vector- database/44249/ pLVX-TetOne-Puro Ciontech clontech.com/US/Products/ Inducible_Systems/TetSystems_ Product_Overview/Tet-One_Overview pMD2.G Addgene addgene.org/12259/ pX330 Addgene addgene.org/vector- database/42230/ pcDNA3.1(+) Addgene addgene.org/vector- database/2093/ pKSV7 Laboratory addgene.org/26686/ of Daniel Portnoy pKSV7-derivative plasmids this paper see Table S2 pPL2oexL Laboratory see FIG. 11 of Daniel Portnoy pPL2oexL-derivative plasmids this paper see Table S2 Sequence-Based Reagents I. GeneBlocks for HEK293T IDT II. see Table S3 expression of phage proteins Software and Algorithms Prism 5 GraphPad graphpad.com/scientific- software/prism/ CRISPRfinder I2BC crispr.i2bc.paris-saclay.fr/Server/ CRISPRDetect Univsersity brownlabtools.otago.ac.nz/ of Otago CRISPRDetect/predict_crispr_array.html CRISPRtarget Univsersity bioanatysis.otago.ac.nz/ of Otago CRISPRTarget/crispr_analysis.html illustrator adobe adobe.com/Illlustrator MEGA6 MEGA megasoftware.net/ Image Lab 5.2.1 BioRad bio-rad.com/en-cn/product/image- lab-software FlowJo FlowJo LLC flowjo.com/

TABLE S2 Strains and Plasmids, strain ID species strain genotype plasmid drug res, (RAU###, pRAU###) 1 Lmo 10403S wt — — 3 E. coli DH5a — pKSV7 amp₁₀₀ 13 Lmo SLCC2482 wt — — 14 Lmo SLCC2540 wt — — 19 Lmo J0161 wt — — 29 E. coli DH5a — pKSV7-S1_(J0161) amp₁₀₀ 31 E. coli DH5a — pKSV7-S1₁₀₄₀₃₆ amp₁₀₀ 57 Lmo 10403S Φ10403S cure (ComK⁺) — — 46 E. coli DB3.1 — pPL2xoeL chlor₃₄ 71 Lmo 10403S ΔComK::ΦJ0161a — — 100 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag6 chlor₃₄ 101 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag7 chlor₃₄ 102 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag9 chlor₃₄ 103 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag10 chlor₃₄ 104 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag5 chlor₃₄ 105 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag3 chlor₃₄ 106 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag1 chlor₃₄ 107 E. coli DH5a — pKSV7-ΔCas9 amp₁₀₀ 109 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag8 chlor₃₄ 111 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag2 chlor₃₄ 112 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU100 — tet₂ 113 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU101 — tet₂ 114 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU102 — tet₂ 115 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU103 — tet₂ 116 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU105 — tet₂ 117 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU106 — tet₂ 118 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU109 — tet₂ 120 E. coli NEB5alpha — pPL2oexL-Cas9 chlor₃₄ 123 E. coli NEB5alpha — pPL2xoeL-ΦJ0161a-frag4 chlor₃₄ 128 Lmo 10403S ComK⁺, ΔtRNA^(Thr)::pRAU104 — tet₂ 130 Lmo 10403S ComK⁺, ΔCas9 — — 142 Lmo 10403S ComK⁺, ΦJ0161 ΔCas9 — — 144 Lmo 10403S ComK⁺, ΔCas9 ΔtRNA^(Arg)::pRAU120 — tet₂ ΔComK::ΦJ0161 ΔCas9 151 Lmo 10403S ΔtRNAArg:pRAU120 — tet₂ 153 E. coli NEB5alpha — pPL2xoeL-LMOG_03145 chlor₃₄ 155 E. coli NEB5alpha — pPL2xoeL-LMOG_03146 chlor₃₄ 157 E. coli NEB5alpha — pPL2xoeL-LMOG_03147 chlor₃₄ 159 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU153 — tet₂ 160 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU155 — tet₂ 161 Lmo 10403S ComK⁺, ΔtRNA^(Arg)::pRAU157 — tet₂ 162 E. coli NEB5alpha — pPL2xoeL-LMOG_03148 chlor₃₄ 165 Lmo 10403S ComK⁺, ΔtRNAArg:pRAU162 — tet₂ 167 E. coli DH5a — pBAD24 amp₁₀₀ 168 E. coli NEB5alpha — pBAD24-LMOG_03146 amp₁₀₀ 171 E. coli NEB5alpha — pBAD24-LMOG_03147 amp₁₀₀ 173 E. coli NEB5alpha — pBAD24-LMOG_03148 amp₁₀₀ 233 E. coli NEB5alpha — pKSV7-ΔLMOG_03146-7 amp₁₀₀ 239 Lmo 10403S ComK⁺, ΔtRNAArg::pCW3 — tet₂ 241 Lmo 10403S ComK⁺, ΔtRNAArg::pCW7 — tet₂ 243 Lmo 10403S ComK⁺, ΔtRNAArg::pCSW9 — tet₂ 246 Lmo 10403S ΔComK::phi_J0161a ΔacrllAl-2 — — 257 Lmo 10403S ComK⁺, tRNAArg::pCSW29 — tet₂ 259 Lmo 10403S ComK⁺, tRNAArg::pCSW33 — tet₂ 260 Lmo 10403S ComK⁺, tRNAArg::pCSW35 — tet₂ (CSW##, pCS##) 3 E. coli NEB5alpha — pPL2oexL- chlor₃₄ Imoslcc2482_0685 7 E. coli NEB5alpha — pPL2oexL- chlor₃₄ Imoslcc2540_1277 9 E. coli NEB5alpha — pPL2oexL- chlor₃₄ LMOG_02993 13 E. coli NEB5alpha — pBAD24- amp₁₀₀ Imoslcc2482_0685 18 E. coli NEB5alpha — pBAD24- amp₁₀₀ Imoslcc2540_1277 21 E. coli NEB5alpha — pBAD24- amp₁₀₀ LMOG_02993 26 E. coli NEB5alpha — pBAD24- amp₁₀₀ Axk13_03345 29 E. coli NEB5alpha — pPL2oexL- chlor₃₄ Imoslcc2482_0688 33 E. coli NEB5alpha — pPL2oexL- chlor₃₄ Imoslcc2540_1278 35 E. coli NEB5alpha — pPL2oexL- chlor₃₄ LMOG_02992 65 E. coli NEB5alpha — pBAD24- amp₁₀₀ Imoslcc2482_0688 (MS##) 101 E. coli BW25113 — — 161 E. coli BW25113 nfsA::mrfp — kan₃₀ 243 E. coli BW25113 nfsA::mrfp, λatt::pCs550-r — kan₃₀,chlor₂₀ 270 E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 — kan₃₀,gent₁₀ 271 E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, — kan₃₀,chlor₂₀,gent₁₀ Tn7att::spy-dcas9 270-262 E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pBAD24 kan₃₀,gent₁₀,amp₁₀₀ 270-168 E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pRAU168 kan₃₀,gent₁₀,amp₁₀₀ 270-171 E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pRAU171 kan₃₀,gent₁₀,amp₁₀₀ 270-173 E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pRAU173 kan₃₀,gent₁₀,amp₁₀₀ 270-13  E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pCSW13 kan₃₀,gent₁₀,amp₁₀₀ 270-18  E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pCSW18 kan₃₀,gent₁₀,amp₁₀₀ 270-21  E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pCSW21 kan₃₀,gent₁₀,amp₁₀₀ 270-26  E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pCSW26 kan₃₀,gent₁₀,amp₁₀₀ 270-65  E. coli BW25113 nfsA::mrfp, Tn7att::spy-dcas9 pCSW29 kan₃₀,gent₁₀,amp₁₀₀ 271-262 E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pBAD24 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-168 E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pRAU168 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-171 E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pRAU171 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-173 E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pRAU173 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-13  E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pCSW13 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-18  E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pCSW18 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-21  E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pCSW21 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-26  E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pCSW26 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ 271-65  E. coli BW25113 nfsA::mrfp, λatt::pCs550-r, pCSW29 kan₃₀,chlor₂₀,gent₁₀, Tn7att::spy-dcas9 amp₁₀₀ by genome 1 alone 2 alone 4 alone 3 alone 1 + 2 1 + 2 + 3 1 + 4 8 2 11 1 28 48 12 by neighborhood 1 + 2 + 3 1 + 2 1 alone 1 + 4 4 alone 2 alone 4 alone 50 29 16 P 11 1 0 all isolated acrIIA3 EXL25968.1 cut off prophage all isolated acrIIA2 KXS56902.1 cut off KXX34219.1 context unknown; prob not phage all isolated acrIIA4 EHY61390.1 arr1a plasmid (FSL J1208) KXS57719.1 arr1a genome KXW85912.1 arr1a genome KXS77365.1 arr1b genome KXS78354.1 arr1b genome KXX11218.1 arr1b genome KXX17138.1 arr1b genome KXX18287.1 arr1b genome KXS56935.1 arr1c genome AEH91120.1 arr2 prophage ACK40885.1 arr2 prophage all isolated acrIIA1 KTA68177.1 upstr-orf5 prophage AGR15693.1 upstr-orf5 prophage AGR27297.1 upstr-orf5 prophage ALU78083.1 upstr-orf5 prophage EEW20426.1 upstr-orf5 prophage EFK41083.1 upstr-orf5 prophage EZH69029.1 contig cut off KHK04755.1 upstr-orf5 KHK19909.1 upstr-orf5 prophage KHK17523.1 upstr-orf5 KTA28092.1 upstr-orf5 KXS64534.1 contig cut off prophage KHK12400.1 upstr-orf5 KKD43688.1 upstr-orf5 KLH0251.1 contig cut off prophage KTA35071.1 upstr-orf5 KTA45326.1 upstr-orf5 KTA50988.1 upstr-orf5 KTA63900.1 upstr-orf5

EXPERIMENTAL MODEL AND SUBJECT DETAILS Microbes.

Listeria monocytogenes strains were cultured on Brain-Heart Infusion (BHI) medium. Escherichia coli strains were cultured on LB medium.

Cell Lines

Human Embryonic Kidney 293 plus T cell antigen (HEK293T, CRL-3216, ATCC) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals) and 50 μg/mL penicillin/streptomycin (P/S, UCSF CCF).

EXAMPLE 2

We sought to identify homologs of acrIIA1-4 (FIG. 13) that possess anti-CRISPR function against the Streptococcus pyogenes ortholog of Cas9, which is widely used for gene editing.

FIG. 14: AcrIIA1 possesses anti-Cas9 activity in a heterologous system (i.e. outside of natural organism). Previously, this protein did not have anti-Cas9 function in E. coli or human cells. The reason for this discrepancy is not yet known, but compared to positive control AcrIIA4, AcrIIA1 functions very well in this heterologous system (P. aeruginosa), while targeting Cas9.

FIG. 15: AcrIIA2 homologs were identified via sequence alignments, to broadly sample the natural sequence space. Three homologs were tested in addition to the original protein, and as shown using phage plaque assays in FIG. 16: AcrIIA2b.1 and AcrIIA2b.3 have strong anti-SpyCas9 activity compared to the original protein, AcrIIA2a.

FIG. 17: Similar homology searches were performed to identify AcrIIA3 homologs, AcrIIA3b.2, 3b.3, 3b.4. As shown in FIG. 18 with an assay where bacteria spotted on a plate, these three new anti-CRISPR proteins are not toxic and work robustly in bacteria. AcrIIA4, as identified in the paper is a strong and broad spectrum Cas9 inhibitor that binds tightly to Cas9 (see Dong et al Nature, and Shin et al Science Advances). One homolog (AcrIIA4b) was identified to have modest anti-Cas9 activity.

FIG. 19 summarizes the results described in Example 2.

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It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

AcrIIA1 protein sequences >WP_003722518.1 (LMOG_03146) SEQ ID NO: 1 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIKKDVCKALENAITVL KEKKNELL >WP_060571535.1 SEQ ID NO: 2 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESTNIEVLPFTFNRFENEEHVNIKKDVCKALENAITVL KEKKNELL >WP_070783094.1 SEQ ID NO: 3 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESNIEVLPFTFNRFENEEHVNIGKDVCKALENAITVLK EKKNELL >WP_031669445.1 SEQ ID NO: 4 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELL >WP_070286809.1 SEQ ID NO: 5 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESNIEVLPFTFNRFENEKHVNIKKDVCKALENAITVLK EKKNELL >WP_070213372.1 SEQ ID NO: 6 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLNKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELL >WP_003731275.1 SEQ ID NO: 7 MAIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEKHVNIKKDVCKALENAITVL KEKKNELL >WP_010989942.1 SEQ ID NO: 8 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELL >WP_070286796.1 SEQ ID NO: 9 MKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSILRS LSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEFEL YCLSKEFESANIEVLPFTFNRFENEKHVNIKKDVCKALENAITVLKE KKNELL >WP_038409766.1 SEQ ID NO: 10 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLNKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELL >WP_060595919.1 SEQ ID NO: 11 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_047934203.1 SEQ ID NO: 12 MTIKILDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKSLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLGKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >YP_009044824.1 SEQ ID NO: 13 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANSEVLTFTFNRFENEEHADIEKDVKKALNNAIAVL KEKKEELL >WP_061396064.1 SEQ ID NO: 14 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKSLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLGKYKLSFPAQEF ELYCLIKEFESANIEVLTFTFNRFENEEHADIEKDVKKALNNAIAVL KEKKEELL >WP_014930689.1 SEQ ID NO: 15 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKSLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLGKYKLSFPAQEF ELYCLIKEFESANIEVLTFTFNRFENEEHADIEKDVKKALNNAIAVL KAKKEELL >WP_061105218.1 SEQ ID NO: 16 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKSLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLGKYKLSFPAQEF ELYCLIKEFESANSEVLTFTFNRFENEEHADIEKDVKKTLNNAIAVL KEKKEELL >WP_070216262.1 SEQ ID NO: 17 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLTFTFNRFENEEHADIEKDVKKALNNAIAVL KEK >WP_070761486.1 SEQ ID NO: 18 MTSKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKSLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLGKYKLSFPAQEF ELYCLIKEFESANIEVLTFTFNRFENEEHADIEKDVKKALNNAIAVL KEKKKNCYKNY >WP_070005110.1 SEQ ID NO: 19 MSIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKSLNKYTVSIL RALALITGMPISDVLFELEDLEKNADDLAGFKHLLDTYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFESETHVDIEKDVRKALENAITVL KEKKNEFM >WP_070784182.1 SEQ ID NO: 20 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKA >WP_070776459.1 SEQ ID NO: 21 MSIKLLDEFLKKHDLTRYGLSKLTGISGNTLKDGNEKTLNKYTVSIL RALALITGMPISDVLFELEDLEKNADDLAGFKHLLDTYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFESETHVDIEKDVQKALENAITVL KEKKNEFM >WP_003737351.1 SEQ ID NO: 22 MSIKLLDEFLKKHDLTRYGLSKLTGISGNTLKDGNEKTLNKYTVSIL RALALITGMPISDVLFELEDLEKNADDLAGFKGLLDTHKLSFPAHEF ELYCLIKEFESVNIEVLPFTFNRFESETHVDIEKDVRKALENAITVL KEKKNEFM >WP_010991654.1 SEQ ID NO: 23 MSIKLLDEFLKKHDLTRYGLSKLTGISGNTLKDGNEKTLNKYTVSIL RALALITGMPISDVLFELEDLEKNADDLAGFKGLLDTHKLSFPAHEF ELYCLIKEFESVNIEVLPFTFNRFESETHVDIEKDVRKALENAITVL KEKKNEFI >WP_070295945.1 SEQ ID NO: 24 MSIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKTLNKYTVSIL RALALITGMPSSDVLFELEDLEKNADDLAGFKQLLDTHKLSFPAHEF ELYCLIKEFESVNIEVLPFTFNRFESETHVDIEKDVQKALENAIAVL KEKKEELL >WP_061662200.1 SEQ ID NO: 25 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKPLNKYTVSIL RSLSLISGLSVSDVLFELEDIEKNSDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVN >WP_061665494.1 SEQ ID NO: 26 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIKKDVCKALENAITVL KEKKNELL >WP_061107167.1 SEQ ID NO: 27 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIKKDVCKALENAITVL KEKKNELL >WP_070005290.1 SEQ ID NO: 28 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELL >WP_039385152.1 SEQ ID NO: 29 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVFIELAELEKSYDDLAGFKHLLNKYKLSFPAQEF ELYCLIKEFDSANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELL >WP_015967154.1 SEQ ID NO: 30 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELI >WP_069001242.1 SEQ ID NO: 31 MNIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVFIELAELEKSHDDLAGFKHLLDKHKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKALENAITVL KEKKNELL >WP_070040173.1 SEQ ID NO: 32 MSIKLLDEFLKKHDLTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVLIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_068999238.1 SEQ ID NO: 33 MTSKLLDEFLKKHSLTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVLIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_070784648.1 SEQ ID NO: 34 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVLIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_070777825.1 SEQ ID NO: 35 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVLIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_047934326.1 SEQ ID NO: 36 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGiSTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_003727802.1 SEQ ID NO: 37 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVFIELAELEKNYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_003723291.1 (LMOG_02992) SEQ ID NO: 38 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGSTFDVFNELEELEKNYDDLAGFKHLLDKYKLSFSAQEFE LYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVLK EKKNELI >WP_069027465.1 SEQ ID NO: 39 MTIKLLDEFLKKHSKTRYGLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_047933338.1 SEQ ID NO: 40 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_060579665.1 SEQ ID NO: 41 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELL >WP_031646274.1 SEQ ID NO: 42 MTIKLLDEFLKKHDLTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHTDIEKDVKKTLNNAIAVL KEKKEELL >WP_070776287.1 SEQ ID NO: 43 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANSEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_031645842.1 SEQ ID NO: 44 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFM RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_046376633.1 SEQ ID NO: 45 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFKELEELEKNYDDLAGFKHLLDKYKLSFSAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_070242402.1 SEQ ID NO: 46 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLSGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_047923954.1 SEQ ID NO: 47 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESASIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_031668927.1 SEQ ID NO: 48 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVFIELAELEKSHDDLAGFKHLLDKHKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_012581438.1 SEQ ID NO: 49 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_031667947.1 SEQ ID NO: 50 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFECANIEVLPFTFNRFENETHVDIEKDIRKALENAITVL KEKKNELI >WP_061107116.1 SEQ ID NO: 51 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYTVSFL RTLSMCVGMSTVDVFIELAELEKNYDDLAGFKHLLDKYKLSFPAQEF ELYCLSKEFESANIEVLPFTFNRFESETHVDIEKDVKKALNNAIAVL KEKKEELL >WP_070783481.1 SEQ ID NO: 52 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALNNAIAVL KEKKEELL >WP_060577773.1 SEQ ID NO: 53 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKYLLDKHKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVL KEKKNELI >WP_039389295.1 SEQ ID NO: 54 MNIKLLDEFLKKHDLTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCTGISTFDVFIELAELEKSYDDLAGFKHLLDKHKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHADIEKDVKKALNNAIAVL KAKKEELL >WP_070295880.1 SEQ ID NO: 55 MSIKLLDEFLKKHDLTRYQLSKLTGISQNTLKDQNEKTLNKYTVSIL RALALITGMPISDVLFELEDLEKNADDLAGFKQLLDTHKLSFPAHEF ELYCLIKEFESVNIEVLPFTFNRFESETHVDIEKDV >WP_061399219.1 SEQ ID NO: 56 TRYQLSKLTGISQNTLNDYNKKELNKYSVSFLRALSMCAGISTFDVF IELAELEKSYDDLAGFKHLLDKYKLSFPAQEFELYCLIKEFESANIE VLPFTFNRFENEEHVNIKKDVCKALENAITVLKEKKNELL >WP_061112070.1 SEQ ID NO: 57 MKINLLDEFLKRHNITRYRLSKLAGISQNTLKDYNEKSLNKYTVSFL RSLSFVTGEDVTDVLIELAELEKGYDDLAGFKYLLDKYKLSFPALEF ELYCIIKEFESANIEISPFTFNRFENETHVDIEKDVKKALQNAVTVL EERKEELL >WP_070779352.1 SEQ ID NO: 58 MKNNLLDTFLKRHDITRYRLSKLAGISQNTLKDYNEKSLNKYTVSLL RSLSFVTGESITDVLLELAEIEKDYDDLAGFKYLLDKYKLSFPALEF ELYCIIKEFESANVEISPFTFNRFENETHADIEKDVKKALNNAITVL KEKKEELL >WP_014930929.1 SEQ ID NO: 59 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKYLLDKHKLSFPTQEF ELYCLIKEFESANIEVLPFTFNRFENETHADIEKDVKKALNNAIAVL EEKKEELL >WP_069001897.1 SEQ ID NO: 60 MKINLLDAFLKRHNITRYRLSKLAGISGNTLKDYNEKSLNKYTVSFL RSLSFVTGEDVTDVLIELAELEKGYDDLAGFKYLLDKYKLAFPALEF ELYCLIKEFEAANIEVSPFTFNRFENETHADIEKDVKKALKNAIIVL KEKKEELL >WP_070784143.1 SEQ ID NO: 61 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVNIEKDVCKA >EFS02359.1 SEQ ID NO: 62 MKINLLDEFLKRHNITRYRLSKLAGISQNTLKDYTEKSLNKYTVSFL RSLSFATGESVTDILLELAELEKDYDDLAGFKYLLDKYKLAFPALEF ELYCLSKEFESANIEISPFTFNRFESETHTDIEKDVKKALQNAVTVL EERKEELL >WP_061128861.1 SEQ ID NO: 63 MSIKLLDEFLKKHSKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RSLSFATGESVTDILLELAELEKDYDDLAGFKYLLDKYKLAFPALEF ELYCLIKEFESANIEISPFTFNRFESETHTDIEKDVKKALQNAVTVL EERKEELL >KUG37233.1 SEQ ID NO: 64 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKYLLDKHKLSFPTQEF ELYCLIKEFESANIEVLPFTFNRFENETHADIEKDVKKALNNAIAVL EEKKRRTVIKTIDYYDYS >WP_049955951.1 SEQ ID NO: 65 MNNFAFITSFNYQQPRYQLSKLTGISQNTLNDYNKKELNKYSVSFLR ALSMCAGiSTFDVLIELAELEKSYDDLAGFKHLLDKYKLSFPAQEFE LYCLIKEFESANIEVLPFTFNRFENETHVDIEKDVRKALENAITVLK EKKNELI >WP_009917642.1 SEQ ID NO: 66 MKTNLLDTFLKRHGITRYRLSKLAGISQNTLKDYTEKSLNKYTVSFL RSLSFVTGEDVTDVLLELAEIENGYDDLAGFKYLLDKYKLSFPALEF ELYCIIKEFESANIEISPFTFNRFENETHADIEKDVKKALKNAVTVL EERKEELL >WP_070777879.1 SEQ ID NO: 67 MNNFAFITSFNYQQPRYQLSKLTGISQNTLNDYNKKELNKYSVSFLR ALSMCAGSSTFDVLIELAELEKSYDDLAGFKHLLDKYKLSFPAQEFE LYCLIKEFECANIEVLPFTFNRFENETHVDIEKDVRKALENAITVLK EKKNELI >WP_061662201.1 SEQ ID NO: 68 MSIKLLDEFLKKHNKTRYQLSKLTGISQNTLNDYNKKELNKYSVSFL RALSMCAGISTFDVFIELAELEKSYDDLAGFKHLLDKYKLSFPAQEF ELYCLIKEFESANIEVLPFTFNRFENEEHVN AcrIIA2 protein sequences >WP_003722517.1 SEQ ID NO: 69 MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSELK >WP_031668925.1 SEQ ID NO: 70 MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSELN >WP_031646276.1 SEQ ID NO: 71 MTITRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSELK >EZH71062.1 SEQ ID NO: 72 KMTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITK NEKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVE TNDIDDLEIATDEDEMKSGNQEIILKSELN >WP_061662199.1 SEQ ID NO: 73 MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSEL >WP_070026783.1 SEQ ID NO: 74 MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSDNQEIILKSELK >WP_068996202.1 SEQ ID NO: 75 MTITRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDGCEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSELK >WP_009928183.1 SEQ ID NO: 76 MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET KDIDDLEIATDEDEMKSDNQEIILKSELK >WP_014930690.1 SEQ ID NO: 77 MTITTAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSELK >WP_061394923.1 SEQ ID NO: 78 MTITTAQRKYNEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET NDIDDLEIATDEDEMKSGNQEIILKSELK >WP_069001241.1 SEQ ID NO: 79 MTITTAQRKYNEAMHEFINMVDDFEESTPDFAKEVLHDCDYVVVTKN EKYAVALCTLSTDECEYDTNLYLDEKLVDYSTVNVNGVTYYINIVET NDIDDLEIATDEDEMKSDNQEIILKSELK >WP_039389299.1 SEQ ID NO: 80 MTITTAQRKYNEAMHEFINMVDDFEESTPDFAKEVLHDCDYVVVTKN EKYAVALCTLSTDECEYDTNLYLDEKLVDYSTVNVNGVTYYINIVET NDIDDLEIATDEDEMKSDNQKIILKSELK >WP_039385155.1 SEQ ID NO: 81 MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVET KDIDDLEIATDEDKEKHDKQEVIIKSELN >WP_003733874.1 SEQ ID NO: 82 MHEFINMVDDFEESTPDFAKEVLHDSDYVVITKNEKYAVALCSLSTD ECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVETNDIDDLEIATDED EMKSGNQEIILKSELK >WP_070294198.1 SEQ ID NO: 83 MTLTRAQKKYAEAMHEFINMVDDFEESKPNFAKEVLHDSDYVVITKN EKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVVT NEDDFKLATDKDKEKHDKQEVIVKSELN >WP_031649390.1 SEQ ID NO: 84 MTLTTAQRKYNEAMHEFINMVDDFEESTPEFSKEVLNDSDYVVITKN EKYAGALCHVSTDECEDGSNLYIDEKLIDYSTLNVGGVTYYINIVER CEDDLEIATDEDKMKSDNQEIILKNELN >EFR93689.1 SEQ ID NO: 85 MVDDFEESTPEFSKEVLNDSDYVVITKNEKYAGALCHVSTDECEDGS NLYIDEKLIDYSTLNVGGVTYYINIVERCEDDLEIATDEDKMKSDNQ EIILKNELN >WP_070295879.1 SEQ ID NO: 86 MTLTTAQKRYYDAMNEFEAITSKKLEQTPEFSQDLLNDSDYLVITKN EAYAVALCMLDDDKLYLDETLVQSTCLDVEGETYYINFVVTNEDDFK LATDEDKEKHDKQEVIVKSELN >WP_070776458.1 SEQ ID NO: 87 MTLTTAQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLVITKN EAYAVALCMLDDDKLYLDETLVQSTCLDVEGETYYINFVVTNEDDFK LATDEDKEKHDKQEVIVKSELN >WP_070005111.1 SEQ ID NO: 88 MTLTTAQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLVITKN EAYAVALCMLDDDKLYLDETLVQSTCLDVEGETYYINFVVTNEDDFK LATDKDKEKHDKQEVIVKSELN >WP_023553814.1 SEQ ID NO: 89 MTITTAQKRYYDAMNEFEAIISKELEQTPAFSQDLLNDSDYLVITKN EAYAVALCMLDDDKLYLDETLVQSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSELN >WP_031645843.1 SEQ ID NO: 90 MTITTAQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLVITKN EAYAVALCMLDDDKLYIDETLVQSTCLDVEGETYYINFVVTNEDDFK LATDKDKEKHDKQEVIIKSELN >WP_014930930.1 SEQ ID NO: 91 MTLTTAQKRYYDAMNEFEAIISKELEQTRAFSQDLLNDSDYLVITKN EAYAVDLCMLDDDKLYLDETLVQSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDRQEVIIKSELN >WP_070783480.1 SEQ ID NO: 92 MTITTAQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLVVTKN EAYAAALCMLDDDKLYLDETLVQSTCLDVEGEIYYINFVVTNEDDFK LATDKDKEKHDKQEVIVKSELN >WP_070040172.1 SEQ ID NO: 93 MTITTAQKRYYDAMNEFEAITSKGLEQTPEFSQDLLNDFDYLVITKN EAYAAALCMLDDEKLYLDETLVHSTRLDIEDDTYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKRELN >WP_012581437.1 SEQ ID NO: 94 MTITTAQKRYYDAMNEFEAIISKELEQTPAFSQDLLNDSDYLVITKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSELN >WP_061107168.1 SEQ ID NO: 95 MTITTAQKRYYDAMNEFEAIISKELEQTPAFSQNLLNDSDYLVITKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSELN >WP_070243058.1 SEQ ID NO: 96 MTITTAQKRYYDAMNEFEAITSKELEQTPAFSQDLLNDSDYLVITKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIVKSELN >WP_015967155.1 SEQ ID NO: 97 MTITTAQKRYYDAMNEFEAIISKELEQTPAFSQDLLNDSDYLVITKN EAYAVALCLLDDDKLYLDETLVHSTRLNIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSEFN >WP_010989941.1 SEQ ID NO: 98 MTLTTAQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLAVTKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSGLN >WP_010991653.1 SEQ ID NO: 99 MTVTTAQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLAVTKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSELN >WP_068996515.1 SEQ ID NO: 100 MTLTTVQKRYYDAMNEFEAITSKELEQTPEFSQDLLNDSDYLAVTKN EAYAVALCLLDDDKLYLDETLVHSTRFDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSELN >WP_047934322.1 SEQ ID NO: 101 MTLTTVQKRYYDAMNEFEAITSKELEQTPEFSQDSLNDSDYLAVTKN EAYAVALCLLDDDKLYLDETLVHSTRFDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSELN >WP_003727803.1 SEQ ID NO: 102 MTITTAQKRYYDAMNEFEAIISKELEQTPAFSQDLLNDSDYLAVTKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSGLN >WP_061114505.1 SEQ ID NO: 103 MTITTAQKRYYDAMNEFEAITSKELEQTPAFSQDLLNDSDYLAVTKN EAYAVALCLLDDDKLYLDETLVHSTRLDIEDETYYINFVVTNEDDFK LATDEDKEKHDKQEVIIKSGLN >EFS02358.1 SEQ ID NO: 104 MAITLSQRKFYEAINEFEEMTENEVVTSPRIPQDYLNDGDYVVITKS ENYALNLCTTNLEGFEDRHFLDEKLIYSTFVETYSGETYYIYITQTA EFDEDDAVEFLATQEQIYEYHKQEEQKTVILKMELS >WP_069001896.1 SEQ ID NO: 105 MAQTEAQKIFYEAINEFEEMTNEEVVTSPRIPQDYLNDGDYVVITKS ENYALNLCTTDLEGFEDRYFLDEKLIYSTSVETYTDETYYIYITQTT EFEEDNAVEFLATQEQIYEYHKQEEQKTVILKMELS >WP_061665680.1 SEQ ID NO: 106 MTTARKKFYQAISEFEAMTGKDVERTPQIADEVLNDAEYIAFTKTEK YALYLCTSNVEGLEDRYFLDEECLDSTFLETEDNETYYIHFLQETEF SEDDNEDELPLATEEQIEAYDKQEELKAVILKKELN >WP_009917643.1 SEQ ID NO: 107 MRTTAQERLDNAINEFEEITNEEVVTSPRIPQDYLNDGDYVVITKSE NYALNLCTTNLEGFEDRHFLDEKLIYSTFVETYSGETYYIYITQTAE FDEDDAVEFLATQEQIYEYHKQEEQKTVILKMELS >WP_061112069.1 SEQ ID NO: 108 MRTTAQERLDNAINEFEEITNEEVVTSPRIPQDYLNDGDYVVITKSE NYALNLCTTNLEGFEDRHFLDEKLIYSTFVETYAGETYYIYITQTAE FDEDDAVEFLATQEQIYEYHKQEEQKTVILKMELS >WP_003745974.1 SEQ ID NO: 109 MRTTAQERLDNAINEFEEITNEEVVTSPLIPQDYLNDGDYVVITKSE NYALNLCTTNLEGFEDRHFLDEKLIYSTFVETYSGETYYIYITQTAE FDEDDAVEFLATQEQIYEYHKQEEQKTVILKMELS AcrIIA3 protein sequences >WP_014930691.1 (Listeria) SEQ ID NO: 110 MFNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_068996201.1 SEQ ID NO: 111 MYNKAEIMKQAWNWFNDSNIWLSDSEWVSYTDKEKSFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_031646277.1 SEQ ID NO: 112 MYNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKGAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSWVACAMKAVKLHNDLFPQTAA >WP_003727804.1 SEQ ID NO: 113 MYNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSLWACAMKAVKLHNDLFPQTAA >WP_070776457.1 SEQ ID NO: 114 MYNKAEIMKQAWNWFNDSNVWLSDIEWISYTDKEKTFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_015455142.1 SEQ ID NO: 115 MYNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSK AKEEVEESKKESKYIAKSEELKAWNWAERKLGLRFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_070005112.1 SEQ ID NO: 116 MFNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKSFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_068996392.1 SEQ ID NO: 117 MYNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSK AKEEVEEFKKESKYIAKSEELKAWNWAERKLGLRFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >CUK89695.1 SEQ ID NO: 118 MKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSKAKEEVEE SKKESKYIAKSEELKAWNWAERKLGLRFNISDDEKFTSVKDETKINF GLSVWACAMKAVKLHNDLFPQTAA >WP_060577772.1 SEQ ID NO: 119 MYNKAEIMKQAWNWFNNSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_070295878.1 SEQ ID NO: 120 MYNKAEIMKQAWNWFNNSNVWLSDIEWVSYTDKEKTFSVCLRAAWSK AKEEVEESKEESKHIAKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_061396065.1 SEQ ID NO: 121 MFNKAEIMKQAWNWFTDSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_039389302.1 SEQ ID NO: 122 MYNKAEIMKQAWNWFNDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_047934319.1 SEQ ID NO: 123 MFNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_069001240.1 SEQ ID NO: 124 MYNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_010991652.1 SEQ ID NO: 125 MFNKAEIMKQAWNWFTDSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEFKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK NETKMNFGLSVWACAMKAVKLHNDLFPQTAA >WP_061114504.1 SEQ ID NO: 126 MYNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTVA >WP_014930931.1 SEQ ID NO: 127 MYNKAEIMKQAWNWFNDSNVWLSDiEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHSHLFPQTAA >WP_069002681.1 SEQ ID NO: 128 MYNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKREELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTVA >WP_012581436.1 SEQ ID NO: 129 MFNKAEIMKQAWNWFTDSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLRFNISDDEKFTSVK DETKQHFGLSVWACAMKAVKLHNDLFPQTAA >WP_010989940.1 SEQ ID NO: 130 MYNKAEIMKQAWNWFNDSNIWLSDIEWVSYTDKEKSFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAESKLGLRFNISDDEKFTSVK DETKMNFDLNVWACAMKAVKLHNDLFPQTAA >WP_015967156.1 SEQ ID NO: 131 MYNKAEIMKQAWNWFTDSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLRFNSSDDEKFTSVK DETKQHFGLSVWACAMKAVKLHNDLFPQTAA >WP_031645844.1 SEQ ID NO: 132 MYNKAEIMKQAWNCFNDSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEIEKSKKESKHIAKSEELKAWNWAERKLGLRFNISDDEKFTSVK DETKQHFGLSVWACAMKAVKLHNDLFPQTAA >WP_031691597.1 SEQ ID NO: 133 MYNKAEIMKQAWNCFNDSNVWLSDIEWVSYTDKEKTFSVCLKAAWSK AKEEIEESKKESKHIAKSEELKAWNWAERKLGLRFNISDDEKFTSVK DETKQHFGLSVWACAMKAVKLHNDLFPQTAA >WP_023553812.1 SEQ ID NO: 134 MYNKSEIMGQAWNWFRDSSVWLSDIEVWSYTDKEKTFSVCLKAAWSK AKEEVEESKKESKHIAKSEELKAWNWAESKLGLRFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTAA >WP_069029656.1 SEQ ID NO: 135 MYNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKREELKAWNLAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHNDLFPQTVA >WP_069001927.1 SEQ ID NO: 136 MFNKAEIMKQAWNWFTDSNVWLSDIEWASYTDKEKTFSVCLKAAWSK AKEEVKEVEKEIKHISKSEELKAWNWAERKLGLHFNISDDEKFTSVK DETKINFGLSVWACAMKAVKLHN >WP_064659125.1 SEQ ID NO: 137 MKKVTYDKSGIMKEAWNLFNNDDITLADFEHLGWMEWKSEKTFALCL KEAWGREKEVVERVNQKFANAETSEEAKAWDWACKKLGVAFEMDAYT KMTNVEDMEKEAWPGTSVWSLAMRAVKLHMEVAA >WP_012678885.1 SEQ ID NO: 138 MRYNKSEIMKNAWAMFNSCNWGAENFKFVSVEEKTFAACLKEAWAEE KEYVEEKIKESANAPKSEEAKAWDWACRKLNANKLQNVEATDKVAWV SEMAKEMWSSNIWAQAIKAVKLHIKLFAA >WP_037595340.1 SEQ ID NO: 139 MKYNKSEIMKNAWTMFNDDNFDTSYYEYATAEVYGQKTFSECLKESW GREKAYQEEKEKRLVDAPKSEEAKAWDWACRKLNVNELQNIDATDKV FYVEGMAKEMWSSNVWAQAIKAVKLHIELFVA >WP_009730539.1 SEQ ID NO: 140 MKKVAYDKSGIMKEAWEMFNRNYQICDFEYADFSGREYFEYASFADC LKEAWAHEKEVVERVNQKYADAETSEEVKAWDWACKKLGVAFEMDAY TKITNVEGMEKEAWPGTSVWSLAMRAVKLHMEVAA >WP_071127625.1 SEQ ID NO: 141 MAKYNKSEIMTQAWTLFNSDNFDTCDYEYTTALVYGQKNFSDCLKEA WGREKAIVERMAEQEANAPLSEEAKAWDWACRKLGVTAEVTAVEKVR YVDDMAKEMWSANVWKQAIKAVQLYATVA >AGM98706.1 SEQ ID NO: 142 MAKYNKSEIMTQAWTLFNSDNFDTCDYEYATALVYGQKTFSDCLKEA WGREKAIVERMAEKEANAPLSEEAKAWDWACRKLGVTAEVTAVEKVR YVDDMAKEMWSTNVWKQAIKAVQLYATVA >WP_012767357.1 SEQ ID NO: 143 MAKYNKSEIMKNAWAMFNSYEWDVENFKFVSAENKTFSNCLKEAWAE EKEYVERKAKETAEAPKSEEAKAWDWACRKLNVNDLQNIDATDKVFY VVDMQKEMWTSNVWAQAIKAVELYVKLGLA >WP_023611744.1 (Streptococcus) SEQ ID NO: 144 MTKYNKSEIMKNAWAMFNSYEWDVENFKFVSAENKTFSNCLKEAWAE EKEYVERKAKETAEAPRSEEAKAWDWACRKLNVNDLQNIDATDKVFY VVDMQKEMWTSNVWAQAIKAVELYVKLGLA >WP_066028552.1 SEQ ID NO: 145 MTKYNKSEIMKNAWAMFNSYEWDVENFKFVSAENKTFSNCLKEAWAE EKEYVERKAKETAEAPKSEEAKAWDWACRKLNVNDLQNIDATDKVFY VVDMQKEMWTSNIWAQAIKAVELYVKLGLA >WP_003055844.1 SEQ ID NO: 146 MTKYNKSEIMKNAWAMFNSYEWDVENFKFVSAENKTFSNCLKEAWAE EKEYVERKAKEAAEASKSEEAKAWDWACRKLNVNDLQNIDATNKVFY VVDMQKEMWTSNWVAQAIKAVELYVKLGLA AcrIIA4 protein sequences >WP_003723290.1 SEQ ID NO: 147 MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISESE NESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN >WP_046376634.1 SEQ ID NO: 148 MNINELIREIKNKDYTAKLSGTDSNSITQLIIRVNNDGNEYVISESE NESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN >WP_069001216.1 SEQ ID NO: 149 MNINELIREVKNKDYTAKLSGTDSNSITQLIIRVNNDGNEYVISESE NESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN >KLI10194.1 SEQ ID NO: 150 MNINELIREIKNKDYTAKLSGTDSNSIAQLIIRVNNDGNEYVISESE NESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN >WP_031667946.1 SEQ ID NO: 151 MNINELIREIKNKDY7AKLSGTDSNSITQLIIHVNNDGNEYVISESE NESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN >WP_070295973.1 SEQ ID NO: 152 MNINDLIREIKNKDYTVKLSGTDSNSITQLIINVNNDGNEYGISESN FESIVEKFVSNFENGWDGAYEDEEEFYNDMQAISLKSELN >WP_061107115.1 SEQ ID NO: 153 MNINDLIREIKNKDYTVKLSGTDSNSITQLIINVNNDGNEYGISESN FESIVEKFVSNFENGWDGAYEDEEEFYNDMQAIILKSESN >WP_060954847.1 SEQ ID NO: 154 MNISELIREIKNKDYTVRLEGTDDNSITKLIIDVDNDGNEYVISESK NESIAEKFASTFKNGWNKEYEDEEEFYNDMQSIILKSELN >WP_012582157.1 SEQ ID NO: 155 MNISELSREIKNKDYAVRLEGTDDNSITKLIIDVDNDGNEYVISESK NESIAEKFASTFKNGWNKEYEDEEEFYNDMQSIILKSELN >CAR82813.1 SEQ ID NO: 156 MAGYLKRYAEDRGWTLYRLAKESHLSDSTLRTADLTTLNKLSVINIK KISEAVGETPGEVLDDLIKFEERVEKMNISELIREIKNKDYAVRLEG TDDNSITKLIIDVDNDGNEYVISESKNESIAEKFASTFKNGWNKEYE DEEEFYNDMQSIILKSELN >WP_003740262.1 SEQ ID NO: 157 MNLKELVREIKNKDYTAKLSGTDSNSITQLIIHVNNDGNEYGISESN FESIVEKFVSTFENGWDGAYEDEEEFYNDMQDIVNRHFK >WP_061385557.1 SEQ ID NO: 158 MNLKELVREIKNKDYTAKLSGTDSNSITQLIIHVNNDGNEYGISESN FESIVEKFVSNFENGWDGAYEDEEEFYNDMQDIVNRHFK >CUL91420.1 SEQ ID NO: 159 MKINELVREIKSRDYTVRLNGTDSNSITKLIIDVNNDGNEYVISERQ DTSIVESFADSFIDGWTGTYEDEEDFYNDMQEIAQDIILETLKEAFE NNNYNTDEVDTDLFDGYQIKLAMEYDNIGELATSVNKTKHFTAYMDA STDFMIIEKY >YP_008239985.1 SEQ ID NO: 160 MSIIAIKKEIHAKGYKVTGTHQGYIAQINFDGTGNEYPLPATWDEFI ETFKDGWNGTYEDEQAFFNDMQEVALKEILDELTGALFCQDITTYDF TIDDVKKKVITLDKPTFEEDAEDLIIEFDSTCFWDATVENDKIKITV RNKSRY >AII27415.1 SEQ ID NO: 161 MSIIAIKKEIHAKGYKVTGTHQGYIAQINFDGTGNEYPLPATWDEFI ETFKDGWNGTYEDEQAFFNDMQEIALDEILDELIDVLYNLDITTYNF TIDDS >YP_001468568.1 SEQ ID NO: 162 MSIIAIKKEIHAKGYKVTGTHQGYIAGINFDGTGNEYPLPATWDEFI ETFKDGWNGTYEDEQAFFNDMQEIALEEILDELTGALFCQDITTYDF TIDDVKKKVITLDKPTFEEDAEDLISEFDSTCFWDATVENDKSKITV RNKSRY >YP_009043548.1 SEQ ID NO: 163 MSIIAINKEIRAKGYKVTGTHQGYIAQINFEGTGNEYPLPATWDEFI ETFKDGWNGTYEDEQAFFNDMQEIALDEILDELIDVLYNLDITTYNF TIDDVKKKVITLNKPIDEEETEDLVQEFNVTCFWDATVEDDKVKVTI RNKNRAIS >AID17477.1 SEQ ID NO: 164 MSTTAINKEIHAKGYKVTGTHQGYIAQINFDGTGNEYPLPATWEKFI ETFKDGWDGTYEDEQAFFNDMQEIALDEILDELIDVLYNLDITTYNF TIDDVKKKVITLNKPIDEEETEDLVQEFNVTCFWNAIVEDDKVKITV RNKSK >YP_009043010.1 SEQ ID NO: 165 MSTTAINKEIHAKGYKVTGTHQGYIAQINFDGTGNEYPLPATWEEFI ETFKDGWDGTYEDEQAFFNDMQEIALDEILDELIDVLYNLDITTYNF TIDDVKKKVITLNKPIDEEETEDLVQEFNVTCFWNAIVEDDKVKITV RNKSK >AID17274.1 SEQ ID NO: 166 MSTTAINKEIHAKGYKVTGTHQGYVAQINFDGTGNEYPLPATWEEFI ETFKDGWDGTYKDEQAFFNDMQEIALDEILDELIDTLYNLDITTYDF TIDDIKKKVITLDKPTDREETEDLVQEFNVTCFWNAIVEDDKVKVTV RNKSK >AAY53411.1 SEQ ID NO: 167 MTGTHQGYIAQINFDGTGNEYPLPATWDEFIETFKDGWNGTYEDEQA FFNDMQEVALKEILDELIDVLYNLDITTYNFTIDDVKKKVITLNKPT DEEDAEDLVIEFDSTCFXDATVENDKIKVTVRNKSK >YP_009044467.1 SEQ ID NO: 168 MSTTAINKEIHAKGYKVTGTHQGYMAQINFDGTGNEFPLPATWEEFI ETFKDGWDGTYEDEQAFFNDMQEVALEELLDELTDVFYNLDITAYDF TVDDVKKKVITLDKPTDREETEDLVQEFKATCFWNAVVEDDKVKVTI RNKNRAIS Additional protein sequences AcrIIA3b.3; OLF47316.1 SEQ ID NO: 169 MQFVVTNKSELFKFAWKIFKANKDIAFSECLQNAWFQYKRYLNREAI KAAQQRKLAKFIADTENEEVKAWNWAEKKLGVALNLTDAEKERNVRN MYKEMWNANVWATAIKAVKLHMEIG AcrIIA4b.2, YP 008240385.1 SEQ ID NO: 170 MNELRSLEMSINAKKYDTRLESGNRVLNIGFGDGEDYPVCSSSRYSL KESFIECFKDGWSGTYRDEKELMEDMQEIAQELILEELTDIFEYYEF NTDEIDTDLFKGFTFDVDSDLEDSMALMKAINATKYFEARSSSWYAS FEVSYIG 

What is claimed is:
 1. A method of inhibiting a Cas9 polypeptide in a cell, the method comprising, introducing a Cas9-inhibiting polypeptide into a cell, wherein: the Cas9-inhibiting polypeptide is heterologous to the cell, and the Cas9-inhibiting polypeptide is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%) identical to any one or more of SEQ ID NO: 1-170; thereby inhibiting the Cas9 polypeptide in a cell.
 2. The method of claim 1, comprising contacting the Cas9 inhibiting polypeptide with a Cas9 polypeptide in the cell.
 3. The method of claim 1, wherein the Cas9-inhibiting polypeptide comprises one of SEQ ID NO: 1-170.
 4. The method of claim 1, wherein the cell comprises the Cas9 polypeptide before the introducing.
 5. The method of claim 4, wherein the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding the Cas9 polypeptide.
 6. The method of claim 5, wherein the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell prior to the introducing.
 7. The method of claim 1, wherein the cell comprises the Cas9 polypeptide after the introducing.
 8. The method of claim 7, wherein the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell after to the introducing.
 9. The method of claim 1, wherein the introducing comprises expressing the Cas9-inhibiting polypeptide in the cell from an expression cassette that is present in the cell and heterologous to the cell, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide.
 10. The method of claim 9, wherein the promoter is an inducible promoter and the introducing comprises contacting the cell with an agent that induces expression of the Cas9-inhibiting polypeptide.
 11. The method of claim 1, wherein the introducing comprises introducing an RNA encoding the Cas9-inhibiting polypeptide into the cell and expressing the Cas9-inhibiting polypeptide in the cell from the RNA.
 12. The method of claim 1, wherein the introducing comprises inserting the Cas9-inhibiting polypeptide into the cell or contacting the cell with the Cas9-inhibiting polypeptide.
 13. The method of any of claims 1-12, wherein the cell is a eukaryotic cell.
 14. The method of claim 13, wherein the cell is a mammalian cell.
 15. The method of claim 14, wherein the cell is a human cell.
 16. The method of any of claims 14-15, wherein the cell is a blood or an induced pluripotent stem cell.
 17. The method of any of claims 14-16, wherein the method occurs ex vivo.
 18. The method of claim 17, wherein the cells are introduced into a mammal after the introducing and contacting.
 19. The method of claim 18, wherein the cells are autologous to the mammal.
 20. The method of any of claims 1-12, wherein the cell is a prokaryotic cell.
 21. A cell comprising a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is heterologous to the cell and the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ IL) NO: 1-170.
 22. The cell of any of claim 21, wherein the cell is a eukaryotic cell.
 23. The method of claim 22, wherein the cell is a mammalian cell.
 24. The method of claim 23, wherein the cell is a human cell.
 25. The method of any of claim 21, wherein the cell is a prokaryotic cell.
 26. A polynucleotide comprising a nucleic acid encoding a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO: 1-170
 27. The polynucleotide of claim 26, comprising an expression cassette, the expression cassette comprising a promoter operably linked to the nucleic acid.
 28. The polynucleotide of claim 27, wherein the promoter is heterologous to the polynucleotide encoding the Cas9-inhibiting polypeptide.
 29. The polynucleotide of claim 27 or 28, wherein the promoter is inducible.
 30. The polynucleotide of claim 26, wherein the polynucleotide is DNA or RNA.
 31. A vector comprising the expression cassette of any of claims 27-29.
 32. The vector of claim 31, wherein the vector is a viral vector.
 33. An isolated Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO:1-170.
 34. A pharmaceutical composition comprising the polynucleotide of any of claims 26-29 or the polypeptide of any of claims
 33. 35. A delivery vehicle comprising the polynucleotide of any of claims 26-30 or the polypeptide of claim
 33. 36. The delivery vehicle of claim 35, wherein the delivery vehicle is a liposome or nanoparticle. 